記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：Journal of Biological Chemistry  

Listeriosis, caused by infection with Listeria monocytogenes,is a severe disease with a high mortality rate. The L. monocytogenes virulence factor, internalin family protein InlA,which binds to the host receptor E-cadherin, is necessary to invade host cells. Here, we isolated two single-domain anti-bodies (V HHs) that bind to InlA with picomolar affinities froman alpaca immune library using the phage display method.These InlA-specific V HHs inhibited the binding of InlA to the extracellular domains of E-cadherin in vitro as shown by bio-physical interaction analysis. Furthermore, we determined thatthe V HHs inhibited the invasion of L. monocytogenes into host cells in culture. High-resolution X-ray structure analyses of the complexes of V HHs with InlA revealed that the V HHs bind tothe same binding site as E-cadherin against InlA. We conclude that these VHHs have the potential for use as drugs to treat listeriosis.

DOI： 10.1016/j.jbc.2023.105254

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その他リンク： https://www.sciencedirect.com/science/article/pii/S0021925823022822?via%3Dihub

記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：Biochimica et Biophysica Acta - Biomembranes  

he fusion peptide (FP) and the Trp-rich membrane proximal external region (MPER) display membrane activity during HIV-1 fusion. These domains are highly conserved in the envelope glycoprotein (Env) and, consequently, antibodies targeting these regions block entry of divergent HIV strains and isolates into target cells. With the aim of recovering concurrent responses against the membrane-active Env domains, we have produced hybrid peptides that connect FP and MPER sequences via flexible aminohexanoic acid tethers, and tested their potential as immunogens. We demonstrate that liposome-based formulations containing FP-MPER hybrid peptides could elicit in rabbits, antibodies with the binding sequence specificity of neutralizing antibodies that engage with the N-terminal MPER sub-region. Determination of the thermodynamic parameters of binding using the Fab 2F5 as an N-terminal MPER antibody model, revealed that the hydrophobic interaction surface for epitope engagement appears to be optimal in the FP-MPER hybrid. In general, our data support: i) the use of liposomes as carriers for membrane active peptides; ii) the capacity of these liposome-based vaccines to focus humoral responses to N-terminal MPER epitopes; and iii) the need to include lipid membranes in immunogens to elicit such specific responses.

DOI： 10.1016/j.bbamem.2023.184235

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その他リンク： https://www.sciencedirect.com/science/article/pii/S0005273623001177?via%3Dihub

記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：Protein Science  

Small molecules that regulate protein–protein interactions can be valuable drugs; however, the development of such small molecules is challenging as the molecule must interfere with an interaction that often involves a large surface area. Herein, we propose that modulating the conformational ensemble of the proteins participating in a given interaction, rather than blocking the interaction by directly binding to the interface, is a relevant strategy for interfering with a protein–protein interaction. In this study, we applied this concept to P-cadherin, a cell surface protein forming homodimers that are essential for cell–cell adhesion in various biological contexts. We first determined the crystal structure of P-cadherin with a small molecule inhibitor whose inhibitory mechanism was unknown. Molecular dynamics simulations suggest that the inhibition of cell adhesion by this small molecule results from modulation of the conformational ensemble of P-cadherin. Our study demonstrates the potential of small molecules altering the conformation ensemble of a protein as inhibitors of biological relevant protein–protein interactions.

DOI： 10.1002/pro.4744

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その他リンク： https://onlinelibrary.wiley.com/doi/full/10.1002/pro.4744

記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：Journal of Biological Chemistry  

Methicillin resistant Staphylococcus aureus, or MRSA, is one of the major causative agents of hospital-acquired infections worldwide. Novel antimicrobial strategies efficient against antibiotic-resistant strains are necessary, and not only against S. aureus. Among those, strategies that aim at blocking or dismantling proteins involved in the acquisition of essential nutrients, helping the bacteria to colonize the host, are intensively studied. A major route for S. aureus to acquire iron from the host organism is the Isd (Iron surface determinant) system. In particular, the hemoglobin receptors IsdH and IsdB located on the surface of the bacterium are necessary to acquire the heme moiety containing iron, making them a plausible antibacterial target. Herein, we obtained an antibody of camelid origin that blocked heme acquisition. We determined that the antibody recognized the heme binding pocket of both IsdH and IsdB with nM order affinity through its second and third complementary determinant regions (CDR). The mechanism explaining the inhibition of acquisition of heme in vitro could be described as a competitive process in which the CDR3 from the antibody blocked the acquisition of heme by the bacterial receptor. Moreover, this antibody markedly reduced the growth of three different pathogenic strains of MRSA. Collectively, our results highlight a mechanism for inhibiting nutrient uptake as an antibacterial strategy against MRSA.

DOI： 10.1016/j.jbc.2023.104927

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その他リンク： https://www.sciencedirect.com/science/article/pii/S0021925823019555?via%3Dihub

記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：Communications Biology  

Antibodies against the carboxy-terminal section of the membrane-proximal external region (C-MPER) of the HIV-1 envelope glycoprotein (Env) are considered as nearly pan-neutralizing. Development of vaccines capable of producing analogous broadly neutralizing antibodies requires deep understanding of the mechanism that underlies C-MPER recognition in membranes. Here, we use the archetypic 10E8 antibody and a variety of biophysical techniques including single-molecule approaches to study the molecular recognition of C-MPER in membrane mimetics. In contrast to the assumption that an interfacial MPER helix embodies the entire C-MPER epitope recognized by 10E8, our data indicate that transmembrane domain (TMD) residues contribute to binding affinity and specificity. Moreover, anchoring to membrane the helical C-MPER epitope through the TMD augments antibody binding affinity and relieves the effects exerted by the interfacial MPER helix on the mechanical stability of the lipid bilayer. These observations support that addition of TMD residues may result in more efficient and stable anti-MPER vaccines.

DOI： 10.1038/s42003-022-04219-6

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その他リンク： https://www.nature.com/articles/s42003-022-04219-6

記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：Journal of Medicinal Chemistry  

The coronavirus disease 2019 (COVID-19) pandemic has necessitated the development of antiviral agents against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). 3C-like protease (3CLpro) is a promising target for COVID-19 treatment. Here, we report a new class of covalent inhibitors of 3CLpro that possess chlorofluoroacetamide (CFA) as a cysteine-reactive warhead. Based on an aza-peptide scaffold, we synthesized a series of CFA derivatives in enantiopure form and evaluated their biochemical efficiency. The data revealed that 8a (YH-6) with the R configuration at the CFA unit strongly blocks SARS-CoV-2 replication in infected cells, and its potency is comparable to that of nirmatrelvir. X-ray structural analysis showed that YH-6 formed a covalent bond with Cys145 at the catalytic center of 3CLpro. The strong antiviral activity and favorable pharmacokinetic properties of YH-6 suggest its potential as a lead compound for the treatment of COVID-19.

DOI： 10.1021/acs.jmedchem.2c01081

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その他リンク： https://pubs.acs.org/doi/10.1021/acs.jmedchem.2c01081

記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：Protein Science  

The camelid single domain antibody, referred to VHH or Nanobody, is consid- ered a versatile tool for various biotechnological and clinical applications because of its favorable biophysical properties. To take advantage of these char- acteristics and for its application in biotechnology and therapy, research on VHH engineering is currently vigorously conducted. To humanize a camelid VHH, we performed complementarity determining region (CDR) grafting using a humanized VHH currently in clinical trials, and investigated the effects of these changes on the biophysical properties of the resulting VHH. The chi- meric VHH exhibited a significant decrease in affinity and thermal stability and a large conformational change in the CDR3. To elucidate the molecular basis for these changes, we performed mutational analyses on the framework regions revealing the contribution of individual residues within the framework region. It is demonstrated that the mutations resulted in the loss of affinity and lower thermal stability, revealing the significance of bulky residues in the vicinity of the CDR3, and the importance of intramolecular interactions between the CDR3 and the framework-2 region. Subsequently, we performed back-mutational analyses on the chimeric VHH. Back-mutations resulted in an increase of the thermal stability and affinity. These data suggested that back- mutations restored the intramolecular interactions, and proper positioning and/or dynamics of the CDR3, resulting in the gain of thermal stability and affinity. These observations revealed the molecular contribution of the frame- work region on VHHs and further designability of the framework region of VHHs without modifying the CDRs.

DOI： 10.1002/pro.4450

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その他リンク： https://onlinelibrary.wiley.com/doi/full/10.1002/pro.4450

記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：International Journal of Molecular Sciences  

Antibody engagement with the membrane-proximal external region (MPER) of the envelope glycoprotein (Env) of HIV-1 constitutes a distinctive molecular recognition phenomenon, the full appreciation of which is crucial for understanding the mechanisms that underlie the broad neutralization of the virus. Recognition of the HIV-1 Env antigen seems to depend on two specific features developed by antibodies with MPER specificity: (i) a large cavity at the antigen-binding site that holds the epitope amphipathic helix; and (ii) a membrane-accommodating Fab surface that engages with viral phospholipids. Thus, besides the main Fab–peptide interaction, molecular recognition of MPER depends on semi-specific (electrostatic and hydrophobic) interactions with membranes and, reportedly, on specific binding to the phospholipid head groups. Here, based on available cryo-EM structures of Fab–Env complexes of the anti-MPER antibody 10E8, we sought to delineate the functional antibody–membrane interface using as the defining criterion the neutralization potency and binding affinity improvements induced by Arg substitutions. This rational, Arg-based mutagenesis strategy revealed the position-dependent contribution of electrostatic inter- actions upon inclusion of Arg-s at the CDR1, CDR2 or FR3 of the Fab light chain. Moreover, the contribution of the most effective Arg-s increased the potency enhancement induced by inclusion of a hydrophobic-at-interface Phe at position 100c of the heavy chain CDR3. In combination, the potency and affinity improvements by Arg residues delineated a protein–membrane interaction site, whose surface and position support a possible mechanism of action for 10E8-induced neutralization. Functional delineation of membrane-interacting patches could open new lines of research to optimize antibodies of therapeutic interest that target integral membrane epitopes.

DOI： 10.3390/ijms231810767

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その他リンク： https://www.mdpi.com/1422-0067/23/18/10767

記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：Protein Science  

Diacylglycerol kinases (DGKs) are multi-domain lipid kinases that modulate the levels of lipid messengers, diacylglycerol, and phosphatidic acid. Recently, increasing attention has been paid to its α isozyme (DGKα) as a potential target for cancer immunotherapy. However, little progress has been made on the structural biology of DGKs, and a detailed understanding of the Ca2+-triggered activation of DGKα, for which the N-terminal domains likely play a critical role, remains unclear. We have recently shown that Ca2+ binding to DGKα-EF induces conformational changes from a protease-susceptible “open” conforma- tion in the apo state to a well-folded one in its holo state. Here, we further studied the structural properties of DGKα N-terminal (RVH and EF) domains using a series of biophysical techniques. We first revealed that the N-terminal RVH domain is a novel Ca2+-binding domain, but the Ca2+-induced conforma- tional changes mainly occur in the EF domain. This was corroborated by NMR experiments showing that the EF domain adopts a molten-globule like struc- ture in the apo state. Further analyses using SEC-SAXS and NMR indicate that the partially unfolded EF domain interacts with RVH domain, likely via hydro- phobic interactions in the absence of Ca2+, and this interaction is modified in the presence of Ca2+. Taken together, these results present novel insights into the structural rearrangement of DGKα N-terminal domains upon binding to Ca2+, which is essential for the activation of the enzyme.

DOI： 10.1002/pro.4365

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その他リンク： https://onlinelibrary.wiley.com/doi/full/10.1002/pro.4365

記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：Journal of Biological Chemistry  

Staphylococcus aureus is a major cause of deadly nosocomial infections, a severe problem fueled by the steady increase of resistant bacteria. The iron surface determinant (Isd) system is a family of proteins that acquire nutritional iron from the host organism, helping the bacterium to proliferate during infection, and therefore represents a promising antibacterial target. In particular, the surface protein IsdH captures hemoglobin (Hb) and acquires the heme moiety containing the iron atom. Structurally, IsdH comprises three distinctive NEAr-iron Transporter (NEAT) domains connected by linker domains. The objective of this study was to characterize the linker region between NEAT2 and NEAT3 from various biophysical viewpoints and thereby advance our understanding of its role in the molecular mechanism of heme extraction. We demonstrate the linker region contributes to the stability of the bound protein, likely influencing the flexibility and orientation of the NEAT3 domain in its interaction with Hb, but only exerts a modest contribution to the affinity of IsdH for heme. Based on these data, we suggest that the flexible nature of the linker facilitates the precise positioning of NEAT3 to acquire heme. In addition, we also found that residues His45 and His89 of Hb located in the heme transfer route toward IsdH do not play a critical role in the transfer rate-determining step. In conclusion, this study clarifies key elements of the mechanism of heme extraction of human Hb by IsdH, providing key insights into the Isd system and other protein systems containing NEAT domains.

DOI： 10.1016/j.jbc.2022.101995

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その他リンク： https://www.sciencedirect.com/science/article/pii/S0021925822004355

記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：Journal of Biological Chemistry  

Atypical hemolytic uremic syndrome (aHUS) is a disease associated with dysregulation of the immune complement system, especially of the alternative pathway (AP). Complement factor H (CFH), consisting of 20 domains called complement control protein (CCP1-20), downregulates the AP as a cofactor for mediating C3 inactivation by complement factor I. However, anomalies related to CFH are known to cause excessive complement activation and cytotoxicity. In aHUS, mutations and the presence of anti-CFH autoantibodies (AAbs) have been reported as plausible causes of CFH dysfunction, and it is known that CFH-related aHUS carries a high probability of end-stage renal disease. Elucidating the detailed functions of CFH at the molecular level will help to understand aHUS pathogenesis. Herein, we used biophysical data to reveal that a heavy-chain antibody fragment, termed VHH4, recognized CFH with high affinity. Hemolytic assays also indicated that VHH4 disrupted the protective function of CFH on sheep erythrocytes. Furthermore, X-ray crystallography revealed that VHH4 recognized the Leu1181–Leu1189CCP20 loop, a known anti-CFH AAbs epitope. We next analyzed the dynamics of the C-terminal region of CFH and showed that the epitopes recognized by anti-CFH AAbs and VHH4 were the most flexible regions in CCP18-20. Finally, we conducted mutation analyses to elucidate the mechanism of VHH4 recognition of CFH and revealed that VHH4 inserts the Trp1183CCP20 residue of CFH into the pocket formed by the complementary determining region 3 loop. These results suggested that anti-CFH AAbs may adopt a similar molecular mechanism to recognize the flexible loop of Leu1181-Leu1189CCP20, leading to aHUS pathogenesis.

DOI： 10.1016/j.jbc.2022.101962

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その他リンク： https://www.jbc.org/article/S0021-9258(22)00402-1/fulltext

記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：Biochemical and Biophysical Research Communications  

AL amyloidosis is a life-threatening disease characterized by the deposition of amyloidogenic immu- noglobulin light chain secreted from clonal plasma cells. Here we established an in-vitro screening system of amyloid inhibition of a variable domain in l6 light chain mutant (Vl6), Wil, and screened a food-additive compound library to identify compounds inhibiting the fibril formation. We found gos- sypetin and isoquercitrin as novel inhibitors. NMR analysis showed that both compounds directly interacted with natively-folded Wil, and proteolysis experiments demonstrated that these compounds conferred proteolytic resistance, suggesting that the compounds enhance the kinetic stability of Wil. Since gossypetin and isoquercitrin specifically interacted with the protein at micromolar concentrations, these compounds could be used as lead to further develop inhibitors against AL amyloidosis.

DOI： 10.1016/j.bbrc.2022.01.066

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その他リンク： https://www.sciencedirect.com/science/article/pii/S0006291X22000869?via%3Dihub

記述言語：英語   掲載種別：研究論文（学術雑誌）  

Diverse genes associated with familial Parkinson’s disease (familial Parkinsonism) have been implicated in mitochondrial quality control. One such gene, PARK7 encodes the protein DJ-1, pathogenic mutations of which trigger its translocation from the cytosol to the mitochondrial matrix. The translocation of steady-state cytosolic proteins like DJ-1 to the mitochondrial matrix upon missense mutations is rare, and the underlying mechanism remains to be elucidated. Here, we show that the protein unfolding associated with various DJ-1 mutations drives its import into the mitochondrial matrix. Increasing the structural stability of these DJ-1 mutants restores cytosolic localization. Mechanistically, we show that a reduction in the structural stability of DJ-1 exposes a cryptic N-terminal mitochondrial- targeting signal (MTS), including Leu10, which promotes DJ-1 import into the mitochondrial matrix for subsequent degradation. Our work describes a novel cellular mechanism for targeting a destabilized cytosolic protein to the mitochondria for degradation.

DOI： 10.1242/jcs.258653

その他リンク： https://journals.biologists.com/jcs/article/134/22/jcs258653/273535/Unfolding-is-the-driving-force-for-mitochondrial

記述言語：英語   掲載種別：研究論文（学術雑誌）  

A substantial body of work has been carried out describing the structural features of the complex between single-domain antibodies (VHHs) and antigens, and the preeminence for epitopes located at concave surfaces of the antigen. However, the thermodynamic basis of binding is far less clear. Here, we have analysed the energetic profiles of five VHHs binding to the catalytic cleft as well as a noncleft epitope of hen egg lysozyme. Various binding energetic profiles with distinctive enthalpic/entropic contributions and structural distribution of critical residues were found in the five antibodies analysed. Collectively, we suggest that from an energetic point of view the binding mechanism is influenced by the shape of the epitope. This information may be beneficial for the design of tailored epitopes for VHHs and their practical use.

DOI： 10.1093/jb/mvab082

その他リンク： https://academic.oup.com/jb/advance-article/doi/10.1093/jb/mvab082/6370158

記述言語：英語   掲載種別：研究論文（学術雑誌）  

Broadly neutralizing antibodies (bnAbs) against HIV-1 are frequently associated with the presence of autoreactivity/polyreactivity, a property that can limit their use as therapeutic agents. The bnAb 4E10, targeting the conserved Membrane proximal external region (MPER) of HIV-1, displays almost pan-neutralizing activity across globally circulating HIV-1 strains but exhibits nonspecific off-target interactions with lipid membranes. The hydrophobic apex of the third complementarity-determining region of the heavy chain (CDRH3) loop, which is essential for viral neutralization, critically contributes to this detrimental effect. Here, we have replaced the aromatic/hydrophobic residues from the apex of the CDRH3 of 4E10 with a single aromatic molecule through chemical modification to generate a variant that preserves the neutralization potency and breadth of 4E10 but with reduced autoreactivity. Collectively, our study suggests that the localized accumulation of aromaticity by chemical modification provides a pathway to ameliorate the adverse effects triggered by the CDRH3 of anti-HIV-1 MPER bnAbs.

DOI： 10.1016/j.isci.2021.102987

その他リンク： https://www.sciencedirect.com/science/article/pii/S258900422100955X

記述言語：英語   掲載種別：研究論文（学術雑誌）  

Liver intestine (LI)-cadherin is a member of the cadherin superfamily, which encompasses a group of Ca2+-dependent cell-adhesion proteins. The expression of LI-cadherin is observed on various types of cells in the human body, such as normal small intestine and colon cells, and gastric cancer cells. Because its expression is not observed on normal gastric cells, LI-cadherin is a promising target for gastric cancer imaging. However, because the cell adhesion mechanism of LI-cadherin has remained unknown, rational design of therapeutic mole- cules targeting this cadherin has been hampered. Here, we have studied the homodimerization mechanism of LI-cadherin. We report the crystal structure of the LI-cadherin homodimer containing its first four extracellular cadherin repeats (EC1-4). The EC1-4 homodimer exhibited a unique architecture different from that of other cadherins reported so far, driven by the interactions between EC2 of one protein chain and EC4 of the second protein chain. The crystal structure also revealed that LI-cadherin possesses a noncanonical calcium ion–free linker between the EC2 and EC3 domains. Various biochem- ical techniques and molecular dynamics simulations were employed to elucidate the mechanism of homodimerization. We also showed that the formation of the homodimer observed in the crystal structure is necessary for LI-cadherin–dependent cell adhesion by performing cell aggregation assays. Taken together, our data provide structural insights necessary to advance the use of LI-cadherin as a target for imaging gastric cancer.

DOI： 10.1016/j.jbc.2021.101054

その他リンク： https://www.sciencedirect.com/science/article/pii/S0021925821008565?via%3Dihub

記述言語：英語   掲載種別：研究論文（学術雑誌）  

The CH2 domain is a critical element of the human Immunoglobulin G (IgG) constant region. Although the CH2 domain is the least stable domain in IgG, it is also a promising scaffold candidate for developing novel therapeutic approaches. Recently, we succeeded in preparing glycosylated and non-glycosylated CH2 domain in the host organism Pichia pastoris. Herein, we verified that glycosylation of the CH2 domain decreased both, its tendency to aggregate and its immunogenicity in mice, suggesting that ag- gregation and immunogenicity are related. In addition, we have produced in P. pastoris a stabilized version of the CH2 domain with and without glycan, and their propensity to aggregate evaluated. We found that stabilization alone significantly decreased the aggregation of the CH2 domain. Moreover, the combination of glycosylation and stabilization completely suppressed its aggregation behavior. Since protein aggregation is related to immunogenicity, the combination of glycosylation and stabilization to eliminate the aggregation behavior of a protein could be a fruitful strategy to generate promising immunoglobulin scaffolds.

DOI： https://doi.org/10.1016/j.bbrc.2021.04.070

その他リンク： https://doi.org/10.1016/j.bbrc.2021.04.070

記述言語：英語   掲載種別：研究論文（学術雑誌）  

Pichia pastoris is a popular eukaryotic system employed for the fast, simple and inexpensive production of recom- binant protein including biotherapeutics such as human al- bumin. The CH2 domain of human Immunoglobulin G (IgG) is a promising scaffold for developing novel thera- peutics. To accelerate the research of CH2 domain, we have established a procedure to highly express human CH2 domain (~150mg/l) as well as human Fc (~30mg/l) in yeast P. pastoris. The procedure yields, simultaneously, a major glycosylated (~70%) and non-glycosylated (~30%) fractions. They can be easily separated with high purity. Although both forms of CH2 domain have essentially the same secondary structure, the pres- ence of the glycan increased the thermal stability of the CH2 domain by about 5C as determined from calorimetry. The purified glycosylated CH2 domain elicited polyclonal antibodies in mouse, recognizing not only the CH2 domain, but also recombinant human Fc and the commer- cial IgG1 antibody Rituxan. Protein A and Protein G binding to the kink region between CH2 domain and CH3 domain of human Fc are used to purify therapeutic proteins. Therefore, these antibodies are candidates to de- velop a novel affinity material to purify human antibodies using their CH2 domain.

DOI： doi:10.1093/jb/mvab039

その他リンク： https://academic.oup.com/jb/advance-article/doi/10.1093/jb/mvab039/6194649

記述言語：英語   掲載種別：研究論文（学術雑誌）  

P2X7 receptors (P2X7Rs) belong to a family of ATP-gated non-selective cation channels. Microglia represent a major cell type expressing P2X7Rs. The activation of microglial P2X7Rs causes the release of pro-inflammatory cytokines such as interleukin-1 (IL-1). This response has been implicated in neuroinflammatory states in the central nervous system and in various diseases, including neuropathic pain. Thus, P2X7R may represent a potential therapeutic target. In the present study, we screened a chemical library of clinically approved drugs (1979 compounds) by high-throughput screening and showed that the Ca2+ channel blocker cilnidipine has an inhibitory effect on rodent and human P2X7R. In primary cultured rat microglial cells, cilnidipine inhibited P2X7R-mediated Ca2+ responses and IL-1 release. Moreover, in a rat model of neuropathic pain, the intrathecal administration of cilnidipine produced a reversal of nerve injury-induced mechanical hypersensitivity, a cardinal symptom of neuropathic pain. These results point to a new inhibitory effect of cilnidipine on microglial P2X7R-mediated inflammatory responses and neuropathic pain, proposing its therapeutic potential.

DOI： https://doi.org/10.3390/cells10020434

その他リンク： https://doi.org/10.3390/cells10020434

記述言語：英語   掲載種別：研究論文（学術雑誌）  

Proteins are modulated by a variety of posttranslational modifications including methylation. Despite its importance, the majority of protein methylation modifications discovered by mass spectrometric analyses are functionally uncharac- terized, partly owing to the difficulty in obtaining reliable methylsite-specific antibodies. To elucidate how functional methylsite-specific antibodies recognize the antigens and lead to the development of a novel method to create such anti- bodies, we use an immunized library paired with phage display to create rabbit monoclonal antibodies recognizing trimethy- lated Lys260 of MAP3K2 as a representative substrate. We isolated several methylsite-specific antibodies that contained unique complementarity determining region sequence. We characterized the mode of antigen recognition by each of these antibodies using structural and biophysical analyses, revealing the molecular details, such as binding affinity toward methyl- ated/nonmethylated antigens and structural motif that is responsible for recognition of the methylated lysine residue, by which each antibody recognized the target antigen. In addition, the comparison with the results of Western blotting analysis suggests a critical antigen recognition mode to generate cross- reactivity to protein and peptide antigen of the antibodies. Computational simulations effectively recapitulated our bio- physical data, capturing the antibodies of differing affinity and specificity. Our exhaustive characterization provides molecular architectures of functional methylsite-specific antibodies and thus should contribute to the development of a general method to generate functional methylsite-specific antibodies by de novo design.

DOI： https://doi.org/10.1074/jbc.RA120.015996

その他リンク： https://doi.org/10.1074/jbc.RA120.015996

記述言語：英語   掲載種別：研究論文（学術雑誌）  

The contribution of membrane interfacial interactions to recognition of membrane-embedded antigens by antibodies is currently unclear. This report demonstrates the optimization of this type of antibodies via chemical modification of regions near the membrane but not directly involved in the recognition of the epitope. Using the HIV-1 antibody 10E8 as a model, linear and polycyclic synthetic aromatic compounds are introduced at selected sites. Molecular dynamics simulations predict the favorable interactions of these synthetic compounds with the viral lipid membrane, where the epitope of the HIV-1 glycoprotein Env is located. Chemical modification of 10E8 with aromatic acetamides facilitates the productive and specific recognition of the native antigen, partially buried in the crowded environment of the viral membrane, resulting in a dramatic increase of its capacity to block viral infection. These observations support the harnessing of interfacial affinity through site-selective chemical modification to optimize the function of antibodies that target membrane-proximal epitopes.

DOI： https://doi.org/10.1016/j.celrep.2020.108037

その他リンク： https://www.sciencedirect.com/science/article/pii/S2211124720310226

記述言語：英語   掲載種別：研究論文（学術雑誌）  

Computer-guided library generation is a plausible strategy to optimize antibodies. Herein, we report the improvement of the affinity of a single-domain camelid antibody for its antigen using such approach. We first conducted experimental and computational alanine scanning to describe the precise energetic profile of the antibody–antigen interaction surface. Based on this characterization, we hypothesized that in-silico mutagenesis could be employed to guide the development of a small library for phage display with the goal of improving the affinity of an antibody for its antigen. Optimized antibody mutants were identified after three rounds of selection, in which an alanine residue at the core of the antibody–antigen interface was substituted by residues with large side- chains, generating diverse kinetic responses, and resulting in greater affinity (>10-fold) for the antigen.

DOI： 10.1093/protein/gzaa006

その他リンク： 10.1093/protein/gzaa006

記述言語：英語   掲載種別：研究論文（学術雑誌）  

Single-domain antibodies (VHHs or nanobodies), developed from heavy chain-only antibodies of camelids, are gaining attention as next-generation therapeutic agents. Despite their small size, the high affinity and specificity displayed by VHHs for antigen molecules rival those of IgGs. How such small antibodies achieve that level of performance? Structural studies have revealed that VHHs tend to recognize concave surfaces of their antigens with high shape-complementarity. However, the energetic contribution of individual residues located at the binding interface has not been addressed in detail, obscuring the actual mechanism by which VHHs target the concave surfaces of proteins. Herein, we show that a VHH specific for hen egg lysozyme, D3-L11, not only displayed the characteristic binding of VHHs to a concave region of the surface of the antigen, but also exhibited a distribution of energetic hot-spots like those of IgGs and conventional protein-protein complexes. The highly preorganized and energetically compact interface of D3-L11 recognizes the concave epitope with high shape complementarity by the classical lock-and-key mechanism. Our results shed light on the fundamental basis by which a particular VHH accommodate to the concave surface of an antigens with high affinity in a specific manner, enriching the mechanistic landscape of VHHs.

DOI： 10.1038/s41598-019-50722-y 11

その他リンク： https://www.nature.com/articles/s41598-019-50722-y

記述言語：英語   掲載種別：研究論文（学術雑誌）  

Oxaliplatin, which is widely used as chemotherapy for certain solid cancers, frequently causes peripheral neuropathy. Commonly described neuropathic symptoms include aberrant sensations such as mechanical allodynia (hypersensitivity to normally innocuous stimuli). Although oxaliplatin neuropathy is a dose‐limiting toxicity, there are no established preventive strategies available at present. By screening several sets of small‐molecule chemical libraries (more than 3,000 compounds in total) using a newly established in vitro high‐throughput phenotypic assay, we identified fulvestrant, a clinically approved drug for the treatment of breast cancer in postmenopausal women, as having a protective effect on oxaliplatin‐induced neuronal damage. Furthermore, histological and behavioural analyses using a rat model of oxaliplatin neuropathy demonstrated the in vivo efficacy of fulvestrant to prevent oxaliplatin‐induced axonal degeneration of the sciatic nerve and mechanical allodynia. Furthermore, fulvestrant did not interfere with oxaliplatin‐induced cytotoxicity against cancer cells. Thus, our findings reveal a previously unrecognised pharmacological effect of fulvestrant to prevent oxaliplatin‐induced painful peripheral neuropathy without impairing its cytotoxicity against cancer cells and may represent a novel prophylactic option for patients receiving oxaliplatin chemotherapy.

DOI： 10.1002/ijc.32043

その他リンク： https://onlinelibrary.wiley.com/doi/full/10.1002/ijc.32043

記述言語：英語   掲載種別：研究論文（学術雑誌）  

Random mutations and selective pressure drive protein adaptation to the changing demands
of the environment. As a consequence, nature favors the evolution of protein diversity. A group of proteins subject to exceptional environmental stress and known for their widespread diversity are the pore-forming hemolytic proteins from sea anemones, known as actinoporins. In this study, we identified and isolated new isoforms of actinoporins from the sea anemone Actinia fragacea (fragaceatoxins). We characterized their hemolytic activity, examined their stability and structure, and performed a comparative analysis of their primary sequence. Sequence alignment reveals that most of the variability among actinoporins is associated with non-functional residues. The di↵erences in the thermal behavior among fragaceatoxins suggest that these variability sites contribute to changes in protein stability. In addition, the protein–protein interaction region showed a very high degree of identity (92%) within fragaceatoxins, but only 25% among all actinoporins examined, suggesting some degree of specificity at the species level. Our findings support the mechanism of evolutionary adaptation in actinoporins and reflect common pathways conducive to protein variability.

DOI： 10.3390/toxins11070401

その他リンク： https://www.mdpi.com/2072-6651/11/7/401

記述言語：英語   掲載種別：研究論文（学術雑誌）  

rreversible inhibition of disease-associated proteins with small molecules is a powerful approach for achieving increased and sustained pharmacological potency. Here, we introduce α-chlorofluoroacetamide (CFA) as a novel warhead of targeted covalent inhibitor (TCI). Despite weak intrinsic reactivity, CFA-appended quinazoline showed high reactivity toward Cys797 of epidermal growth factor receptor (EGFR). In cells, CFA-quinazoline showed higher target specificity for EGFR than the corresponding Michael acceptors in a wide concentration range (0.1–10 μM). The cysteine adduct of the CFA derivative was susceptible to hydrolysis and reversibly yielded intact thiol but was stable in solvent-sequestered ATP-binding pocket of EGFR. This environment-dependent hydrolysis can potentially reduce off-target protein modification by CFA-based drugs. Oral administration of CFA quinazoline NS-062 significantly suppressed tumor growth in a mouse xenograft model. Further, CFA-appended pyrazolopyrimidine irreversibly inhibited Bruton’s tyrosine kinase with higher target specificity. These results demonstrate the utility of CFA as a new class warheads for TCI.

DOI： 10.1038/s41589-018-0204-3

その他リンク： https://www.nature.com/articles/s41589-018-0204-3

記述言語：英語   掲載種別：研究論文（学術雑誌）  

DJ-1 is a Parkinson’s disease associated protein endowed with enzymatic, redox sensing, regulatory, chaperoning, and neuroprotective activities. Although DJ-1 has been vigorously studied for the past decade and a half, its exact role in the progression of the disease remains uncertain. In addition, little is known about the spatiotemporal regulation of DJ-1, or the biochemical basis explaining its numerous biological functions. Progress has been hampered by the lack of inhibitors with precisely known mechanisms of action. Herein, we have employed biophysical methodologies and X-ray crystallography to identify and to optimize a family of compounds inactivating the critical Cys106 residue of human DJ-1. We demonstrate these compounds are potent inhibitors of various activities of DJ-1 in vitro and in cell-based assays. This study reports a new family of DJ-1 inhibitors with a defined mechanism of action, and contributes toward the understanding of the biological function of DJ-1.

DOI： 10.1021/acschembio.8b00701

その他リンク： https://pubs.acs.org/doi/10.1021/acschembio.8b00701

記述言語：英語   掲載種別：研究論文（学術雑誌）  

Molecular recognition is a fundamental event at the core of essentially every biological process. In particular, intermolecular H-bonds have been recognized as key stabilizing forces in antibody-antigen interactions resulting in exquisite specificity and high affinity. Although equally abundant, the role of intramolecular H-bonds is far less clear and not universally acknowledged. Herein, we have carried out a molecular-level study to dissect the contribution of intramolecular H-bonds in a flexible peptide for the recognition by an antibody. We show that intramolecular H-bonds may have a profound, multifaceted and favorable effect on the binding affinity by up to 2kcal mol-1 of free energy. Collectively, our results suggest that anti- bodies are fine tuned to recognize transiently stabilized structures of flexible peptides in solution, for which intramolecular H-bonds play a key role.

DOI： 10.1093/jb/mvy032

その他リンク： https://academic.oup.com/jb/article/164/1/65/4904556

記述言語：英語   掲載種別：研究論文（学術雑誌）  

Protein tyrosine sulfation (PTS) is a post-translational modification regulating numerous biological events. PTS generally occurs at flexible regions of proteins, enhancing intermolecular interactions between proteins. Because of the high flexibility associated with the regions where PTS is generally encountered, an atomic-level understanding has been difficult to achieve by X-ray crystallography or nuclear magnetic resonance techniques. In this study, we focused on the conformational behavior of a flexible sulfated peptide and its interaction with an antibody. Molecular dynamics simulations and thermodynamic analysis indicated that PTS reduced the main-chain fluctuations upon the appearance of sulfate-mediated intramolecular H-bonds. Collectively, our data suggested that one of the mechanisms by which PTS may enhance protein–protein interactions consists of the limitation of conformational dynamics in the unbound state, thus reducing the loss of entropy upon binding and boosting the affinity for its partner.

DOI： 10.1021/acs.biochem.8b00592

その他リンク： https://pubs.acs.org/doi/10.1021/acs.biochem.8b00592

記述言語：英語   掲載種別：研究論文（学術雑誌）  

Small leucine-rich repeat proteoglycan (SLRP) proteins have an important role in the organization of the extracellular matrix, especially in the formation of collagen fibrils. However, the mechanism governing the shape of collagen fibrils is poorly understood. Here, we report that the protein Osteomodulin (OMD) of the SLRP family is a monomeric protein in solution that interacts with type-I collagen. This interaction is dominated by weak electrostatic forces employing negatively charged residues of OMD, in particular Glu284 and Glu303, and controlled by entropic factors. The protein OMD establishes a fast-binding equilibrium with collagen, where OMD may engage not only with individual collagen molecules, but also with the growing fibrils. This weak electrostatic interaction is carefully balanced so it modulates the shape of the fibrils without compromising their viability.

DOI： 10.1038/s42003-018-0038-2

その他リンク： https://www.nature.com/articles/s42003-018-0038-2

記述言語：英語   掲載種別：研究論文（学術雑誌）  

The N-glycan moiety of IgG-Fc has a significant impact on multifaceted properties of antibodies
such as in their effector function, structure, and stability. Numerous studies have been devoted to understanding its biological effect since the exact composition of the Fc N-glycan modulates the magnitude of effector functions such as the antibody-dependent cell mediated cytotoxicity (ADCC), and the complement-dependent cytotoxicity (CDC). To date, systematic analyses of the properties and influence of glycan variants have been of great interest. Understanding the principles on how N-glycosylation modulates those properties is important for the molecular design, manufacturing, process optimization, and quality control of therapeutic antibodies. In this study, we have separated a model therapeutic antibody into three fractions according to the composition of the N-glycan by using a novel FcγRIIIa chromatography column. Notably, Fc galactosylation was a major factor influencing the affinity of IgG-Fc to the FcγRIIIa immobilized on the column. Each antibody fraction was employed for structural, biological, and physicochemical analysis, illustrating the mechanism by which galactose modulates the affinity to FcγRIIIa. In addition, we discuss the benefits of the FcγRIIIa chromatography column to assess the heterogeneity of the N-glycan.

DOI： 10.1038/s41598-018-22199-8

その他リンク： https://www.nature.com/articles/s41598-018-22199-8

記述言語：英語   掲載種別：研究論文（学術雑誌）  

Pore-forming toxins (PFTs) are proteins endowed with metamorphic proper- ties that enable them to stably fold in water solutions as well as in cellular membranes. PFTs produce lytic pores on the plasma membranes of target cells conducive to lesions, playing key roles in the defensive and offensive molecular systems of living organisms. Actinoporins are a family of potent haemolytic toxins produced by sea anemones vigorously studied as a para- digm of a-helical PFTs, in the context of lipid–protein interactions, and in connection with nanopore technologies. We have recently reported that fragaceatoxin C (FraC), an actinoporin, engages biological membranes with a large adhesive motif allowing the simultaneous attachment of up to four lipid molecules prior to pore formation. Since actinoporins also interact with carbohydrates, we sought to understand the molecular and energetic basis of glycan recognition by FraC. By employing structural and biophysical methodologies, we show that FraC engages glycans with low affinity using its lipid-binding module. Contrary to other PFTs requiring separate domains for glycan and lipid recognition, the small single-domain actinoporins economize resources by achieving dual recognition with a single binding module. This mechanism could enhance the recruitment of actinoporins to the surface of target tissues in their marine environment.

DOI： 10.1098/rstb.2016.0216

その他リンク： http://rstb.royalsocietypublishing.org/content/372/1726/20160216

記述言語：英語   掲載種別：研究論文（学術雑誌）  

The exceptional breadth of broadly neutralizing antibodies (bNAbs) against the membrane-proximal external region (MPER) of the transmembrane protein gp41 makes this class of antibodies an ideal model to design HIV vaccines. From a practical point of view, however, the preparation of vaccines eliciting bNAbs is still a major roadblock that limits their clinical application. Fresh mechanistic insights are necessary to develop more effective strategies. In particular, the function of the unusually long complementarity-determining region three of the heavy chain (CDRH3) of 4E10, an anti-MPER bNAb, is an open question that fascinates researchers in the field. Residues comprising the apex region are dispensable for engagement of the epitope in solution; still, their single mutation profoundly impairs the neutralization capabilities of the antibody. Since this region is very hydrophobic, it has been proposed that the apex is essential for anchoring the antibody to the viral membrane where MPER resides. Herein, we have critically examined this idea using structural, biophysical, biochemical, and cell-based approaches. Our results demonstrate that the apex region is not just a “greasy” spot merely increasing the affinity of the antibody for the membrane. We demonstrate the three-dimensional engagement of the apex region of the CDRH3 with the conglomerate of gp41 epitope and membrane lipids as a means of effective binding and neutralization of the virus. This mechanism of recognition suggests a standard route of antibody ontogeny. Therefore, we need to focus our efforts on recreating a more realistic MPER/lipid immunogen in order to generate more effective anti-HIV-1 vaccines.

DOI： 10.1016/j.jmb.2017.03.008

その他リンク： http://www.sciencedirect.com/science/article/pii/S0022283617301122

記述言語：英語   掲載種別：研究論文（学術雑誌）  

The 4E10 antibody displays an extreme breadth of HIV-1 neutralization and therefore constitutes a suitable model system for structure- guided vaccine design and immunotherapeutics against AIDS. In this regard, the relevance of auto- reactivity with membrane lipids for the biological function of this antibody is still a subject of controversy. To address this dispute, herein we have compared the membrane-partitioning ability of the 4E10 antibody and several of its variants, which were mutated at the region of the paratope surface in contact with the membrane-interface. We first employed a physical separation approach (vesicle flotation), and subsequently carried out quantitative fluorescence measurements in an intact system (spectroscopic titration), using 4E10 Fab labeled with the polarity-sensitive 4-Chloro-7- Nitrobenz-2-Oxa-1,3-Diazole (NBD) probe. Moreover, recognition of epitope peptide in membrane was demonstrated by photo-cross-linking assays using a Fab that incorporated the genetically encoded unnatural amino acid p-benzoylphenylalanine (pBPA). The experimental data ruled out that the proposed stereospecific recognition of viral lipids was necessary for the function of the antibody. In contrast, our data suggest that nonspecific electrostatic interactionsbetween basic residues of 4E10 and acidic phospholipids in the membranes contribute to the observed biological function. Moreover, the energetics of membrane-partitioning indicated that 4E10 behaves as a peripheral membrane protein, tightening the binding to the ligand epitope inserted in the viral membrane. The implications of these findings for the natural production and biological function of this antibody are discussed.

DOI： 10.1074/jbc.M117.775429

その他リンク： http://www.jbc.org/content/292/13/5571.long

記述言語：英語   掲載種別：研究論文（学術雑誌）  

Human P-cadherin is a cell adhesion protein of the family of classical cadherins, the overexpression of which is correlated with poor prognosis in various types of cancer. Antibodies inhibiting cell-cell adhesion mediated by P-cadherin show clear therapeutic effect, although the mechanistic basis explaining their effectiveness is still unclear. Based on structural, physicochemical, and functional analyses, we have elucidated the molecular mechanism of disruption of cell adhesion by antibodies targeting human P-cadherin. Herein we have studied three different antibodies, TSP5, TSP7, and TSP11, each recognizing a different epitope on the surface of the cell-adhesive domain (EC1). Although all these three antibodies recognized human P-cadherin with high affinity, only TSP7 disrupted cell adhesion. Notably, we demonstrated that TSP7 abolishes cell adhesion by disabling the so-called X-dimer (a kinetic adhesive intermediate), in addition to disrupting the strand-swap dimer (the final thermodynamic state). The inhibition of the X-dimer was crucial for the overall inhibitory effect, raising the therapeutic value of a kinetic intermediary not only for preventing, but also for reversing, cell adhesion mediated by a member of the classical cadherin family. These findings should help to design more innovative and effective therapeutic solutions targeting human P-cadherin.

DOI： 10.1038/srep39518

その他リンク： http://www.nature.com/articles/srep39518

記述言語：英語   掲載種別：研究論文（学術雑誌）  

DOI： 10.1021/acs.biochem.6b01007

その他リンク： http://pubs.acs.org/doi/abs/10.1021/acs.biochem.6b01007

記述言語：英語   掲載種別：研究論文（学術雑誌）  

The mechanism by which the HIV-1 MPER epitope is recognized by the potent neutralizing antibody 10E8 at membrane interfaces remains poorly understood. To solve this problem, we have optimized a 10E8 peptide epitope and analyzed the structure and binding activities of the antibody in membrane and membrane-like environments. The X-ray crystal structure of the Fab-peptide complex in detergents revealed for the first time that the epitope of 10E8 comprises a continuous helix spanning the gp41 MPER/transmembrane domain junction (MPER-N-TMD; Env residues 671–687). The MPER-N-TMD helix projects beyond the tip of the heavy-chain complementarity determining region 3 loop, indicating that the antibody sits parallel to the plane of the membrane in binding the native epitope. Biophysical, biochemical and mutational analyses demonstrated that strengthening the affinity of 10E8 for the TMD helix in a membrane environment, correlated with its neutralizing potency. Our research clarifies the molecular mechanisms underlying broad neutralization of HIV-1 by 10E8, and the structure of its natural epitope. The conclusions of our research will guide future vaccine-design strategies targeting MPER.

DOI： 10.1038/srep38177

その他リンク： http://www.nature.com/articles/srep38177

記述言語：英語   掲載種別：研究論文（学術雑誌）  

Pore-forming toxins (PFTs) are cytolytic proteins belonging to the molecular warfare apparatus of living organisms. The assembly of the functional transmembrane pore requires several intermediate steps ranging from a water-soluble monomeric species to the multimeric ensemble inserted in the cell membrane. The non-lytic oligomeric intermediate known as prepore plays an essential role in the mechanism of insertion of the class of beta-PFTs. However, in the class of alpha-PFTs, like the actinoporins produced by sea anemones, evidence of membrane-bound prepores is still lacking. We have employed single-particle cryo-electron microscopy (cryo-EM) and atomic force microscopy to identify, for the first time, a prepore species of the actinoporin fragaceatoxin C bound to lipid vesicles. The size of the prepore coincides with that of the functional pore, except for the transmembrane region, which is absent in the prepore. Biochemical assays indicated that, in the prepore species, the N terminus is not inserted in the bilayer but is exposed to the aqueous solution. Our study reveals the structure of the prepore in actinoporins and highlights the role of structural intermediates for the formation of cytolytic pores by an alpha-PFT.

DOI： 10.1074/jbc.M116.734053

その他リンク： http://www.jbc.org/content/291/37/19210.long

記述言語：英語   掲載種別：研究論文（学術雑誌）  

Orderly assembly of classical cadherins governs cell adhesion and tissue maintenance. A key event is the strand-swap dimerization of the extracellular ectodomains of two cadherin molecules from apposing cells. Here we have determined crystal structures of P-cadherin in six different conformational states to elaborate a motion picture of its adhesive dimerization at the atomic level. The snapshots revealed that cell-adhesive dimerization is facilitated by several intermediate states collectively termed X-dimer in analogy to other classical cadherins. Based on previous studies and on the combined structural, kinetic, thermodynamic, biochemical, and cellular data reported herein, we propose that the adhesive dimerization of human P-cadherin is achieved by a stepwise mechanism analogous to that of assembly chaperones. This mechanism, applicable to type I classical cadherins, confers high specificity and fast association rates. We expect these findings to guide innovative therapeutic approaches targeting P-cadherin in cancer.

DOI： 10.1016/j.str.2016.07.002

その他リンク： http://www.cell.com/structure/comments/S0969-2126%2816%2930186-1

記述言語：英語   掲載種別：研究論文（学術雑誌）  

Heme oxygenase (HO) is a ubiquitous enzyme with key roles in inflammation, cell signaling, heme disposal, and iron acquisition. HO catalyzes the oxidative conversion of heme to biliverdin (BV) using a conserved histidine to coordinate the iron atom of bound heme. This His−heme interaction has been regarded as being essential for enzyme activity, because His-to-Ala mutants fail to convert heme to biliverdin in vitro. We probed a panel of proximal His mutants of cyanobacterial, human, and plant HO enzymes using a live-cell activity assay based on heterologous co-expression in Escherichia coli of each HO mutant and a fluorescent biliverdin biosensor. In contrast to in vitro studies with purified proteins, we observed that multiple HO mutants retained significant activity within the intracellular environment of bacteria. X-ray crystallographic structures of human HO1 H25R with bound heme and additional functional studies suggest that HO mutant activity inside these cells does not involve heme ligation by a proximal amino acid. Our study reveals unexpected plasticity in the active site binding interactions with heme that can support HO activity within cells, suggests important contributions by the surrounding active site environment to HO catalysis, and can guide efforts to understand the evolution and divergence of HO function.

DOI： 10.1021/acs.biochem.6b00562

その他リンク： http://pubs.acs.org/doi/abs/10.1021/acs.biochem.6b00562

記述言語：英語   掲載種別：研究論文（学術雑誌）  

The 4E10 antibody recognizes the membrane-proximal external region (MPER) of the HIV-1 Env glycoprotein gp41 transmembrane subunit, exhibiting one of the broadest neutralizing activities known to date. The neutralizing activity of 4E10 requires solvent-exposed hydrophobic residues at the apex of the complementarity-determining region (CDR) H3 loop, but the molecular basis for this requirement has not been clarified. Here, we report the cocrystal structures and the energetic parameters of binding of a peptide bearing the 4E10-epitope sequence (4E10ep) to nonneutralizing versions of the 4E10 Fab. Nonneutralizing Fabs were obtained by shortening and decreasing the hydrophobicity of the CDR-H3 loop (termed delta-Loop) or by substituting the two tryptophan residues of the CDR-H3 apex with Asp residues (termed WDWD), which also decreases hydrophobicity but preserves the length of the loop. The analysis was complemented by the first crystal structure of the 4E10 Fab in its ligand-free state. Collectively, the data ruled out major conformational changes of CDR-H3 at any stage during the binding process (equilibrium or transition state). Although these mutations did not impact the affinity of wild-type Fab for the 4E10ep in solution, the two nonneutralizing versions of 4E10 were deficient in binding to MPER inserted in the plasma membrane (mimicking the environment faced by the antibody in vivo). The conclusions of our structure-function analysis strengthen the idea that to exert effective neutralization, the hydrophobic apex of the solvent-exposed CDR-H3 loop must recognize an antigenic structure more complex than just the linear alpha-helical epitope and likely constrained by the viral membrane lipids.

DOI： 10.1128/JVI.01793-15

その他リンク： http://jvi.asm.org/content/89/23/11975.long

記述言語：英語   掲載種別：研究論文（学術雑誌）  

The bidirectional transformation of a protein between its native water-soluble and integral transmembrane conformations is demonstrated for FraC, a hemolytic protein of the family of pore-forming toxins. In the presence of biological membranes, the water-soluble conformation of FraC undergoes a remarkable structural reorganization generating cytolytic transmembrane nanopores conducive to cell death. So far, the reverse transformation from the native transmembrane conformation to the native water-soluble conformation has not been reported. We describe the use of detergents with different physicochemical properties to achieve the spontaneous conversion of transmembrane pores of FraC back into the initial water-soluble state. Thermodynamic and kinetic stability data suggest that specific detergents cause an asymmetric change in the energy landscape of the protein, allowing the bidirectional transformation of a membrane protein.

DOI： 10.1021/acs.biochem.5b01112

その他リンク： http://pubs.acs.org/doi/10.1021/acs.biochem.5b01112

記述言語：英語   掲載種別：研究論文（学術雑誌）  

The rapid spread of antibiotic-resistance among pathogenic bacteria poses a serious risk for public health. The search for novel therapeutic strategies and antimicrobial compounds is needed to ameliorate this menace. The bifunctional metalloenzyme CapF is an antibacterial target produced by certain pathogenic bacteria essential in the biosynthetic route of capsular polysaccharide, a mucous layer on the surface of bacterium that facilitates immune evasion and infection. We report the first inhibitor of CapF from Staphylococcus aureus, which was identified by employing fragment-based methodologies. The hit compound 3-isopropenyl-tropolone inhibits the first reaction catalyzed by CapF, disrupting the synthesis of a key precursor of capsular polysaccharide. Isothermal titration calorimetry demonstrates that 3-isopropenyl-tropolone binds tightly (KD = 27 ± 7 μM) to the cupin domain of CapF. In addition, the crystal structure of the enzyme-inhibitor complex shows that the compound engages the essential Zn2+ ion necessary for the first reaction catalyzed by the enzyme, explaining its inhibitory effect. Moreover, the tropolone compound alters the coordination sphere of the metal, leading to the overall destabilization of the enzyme. We propose 3-isopropenyl-tropolone as a precursor to develop stronger inhibitors for this family of enzymes to impair the synthesis of capsular polysaccharide in Staphylococcus aureus.

DOI： 10.1038/srep15337

その他リンク： http://www.nature.com/articles/srep15337

記述言語：英語   掲載種別：研究論文（学術雑誌）  

Actinoporins are pore-forming toxins produced by different sea anemones that self-assemble within the membranes of their target cells and compromise their function as a permeability barrier. The recently published three-dimensional structures of two oligomeric complexes formed by fragaceatoxin C point to Val60 as a key residue involved in the oligomerization of the functional pore.
To gain insight into the mechanism of toxin oligomerization, different point mutations have been introduced at this position. Functional characterization of the muteins suggests that Val60 represents a hot-spot where the introduction of mutations hinders protein assembly and reduces the overall affinity for membranes.

DOI： 10.1016/j.febslet.2015.06.012

その他リンク： http://onlinelibrary.wiley.com/doi/10.1016/j.febslet.2015.06.012/abstract

記述言語：英語   掲載種別：研究論文（学術雑誌）  

The membrane-proximal external region (MPER) C-terminal segment and the transmembrane domain (TMD) of gp41 are involved in HIV-1 envelope glycoprotein-mediated fusion and modulation of immune responses during viral infection. However, the atomic structure of this functional region remains unsolved. Here, based on the high resolution NMR data obtained for peptides spanning the C-terminal segment of MPER and the TMD, we report two main findings: (i) the conformational variability of the TMD helix at a membrane-buried position; and (ii) the existence of an uninterrupted alpha-helix spanning MPER and the N-terminal region of the TMD. Thus, our structural data provide evidence for the bipartite organization of TMD predicted by previous molecular dynamics simulations and functional studies, but they do not support the breaking of the helix at Lys-683, as was suggested by some models to mark the initiation of the TMD anchor. Antibody binding energetics examined with isothermal titration calorimetry and humoral responses elicited in rabbits by peptide-based vaccines further support the relevance of a continuous MPER-TMD helix for immune recognition. We conclude that the transmembrane anchor of HIV-1 envelope is composed of two distinct sub-domains: 1) an immunogenic helix at the N terminus also involved in promoting membrane fusion; and 2) an immunosuppressive helix at the C terminus, which might also contribute to the late stages of the fusion process. The unprecedented high resolution structural data reported here may guide future vaccine and inhibitor developments.

DOI： 10.1074/jbc.M115.644351

その他リンク： http://www.jbc.org/content/290/21/12999.long

記述言語：英語   掲載種別：研究論文（学術雑誌）  

Cell-surface Fcgamma receptors mediate innate and adaptive immune responses. Human Fcgamma receptor I (hFcgammaRI) binds IgGs with high affinity and is the only Fcgamma receptor that can effectively capture monomeric IgGs. However, the molecular basis of hFcgammmaRI’s interaction with Fc has not been determined, limiting our understanding of this major immune receptor. Here we report the crystal structure of a complex between hFcgammaRI and human Fc, at 1.80Å resolution, revealing an unique hydrophobic pocket at the surface of hFcgammaRI perfectly suited for residue Leu235 of Fc, which explains the high affinity of this complex. Structural, kinetic and thermodynamic data demonstrate that the binding mechanism is governed by a combination of non-covalent interactions, bridging water molecules and the dynamic features of Fc. In addition, the hinge region of hFcgammaRI-bound Fc adopts a straight conformation, potentially orienting the Fab moiety. These findings will stimulate the development of novel therapeutic strategies involving hFcgammaRI.

DOI： 10.1038/ncomms7866

その他リンク： http://www.nature.com/articles/ncomms7866

記述言語：英語   掲載種別：研究論文（学術雑誌）  

The physicochemical landscape of the bilayer modulates membrane protein function. Actinoporins are a family of potent hemolytic proteins from sea anemones acting at the membrane level. This family of cytolysins preferentially binds to target membranes containing sphingomyelin, where they form lytic pores giving rise to cell death. Although the cytolytic activity of the actinoporin fragaceatoxin C (FraC) is sensitive to vesicles made of various lipid compositions, it is far from clear how this toxin adjusts its mechanism of action to a broad range of physio-chemical landscapes. Herein, we show that the conserved residue Phe-16 of FraC is critical for pore formation in cholesterol- rich membranes such as those of red blood cells. The interaction of a panel of muteins of Phe-16 with model membranes com- posed of raft-like lipid domains is inactivated in cholesterol-rich membranes but not in cholesterol-depleted membranes. These results indicate that actinoporins recognize different membrane environments, resulting in a wider repertoire of susceptible target membranes (and preys) for sea anemones. In addition, this study has unveiled promising candidates for the development of protein-based biosensors highly sensitive to the concentration of cholesterol within the membrane.

DOI： 10.1074/jbc.M114.615211

その他リンク： http://www.jbc.org/content/early/2015/03/10/jbc.M114.615211

記述言語：英語   掲載種別：研究論文（学術雑誌）  

Pore-forming toxins (PFT) are water-soluble proteins that possess the remarkable ability to self-assemble on the membrane of target cells, where they form pores causing cell damage. Here, we elucidate the mechanism of action of the haemolytic protein fragaceatoxin C (FraC), a alpha-barrel PFT, by determining the crystal structures of FraC at four different stages of the lytic mechanism, namely the water-soluble state, the monomeric lipid-bound form, an assembly intermediate and the fully assembled transmembrane pore. The structure of the transmembrane pore exhibits a unique architecture composed of both protein and lipids, with some of the lipids lining the pore wall, acting as assembly cofactors. The pore also exhibits lateral fenestrations that expose the hydrophobic core of the membrane to the aqueous environment. The incorporation of lipids from the target membrane within the structure of the pore provides a membrane-specific trigger for the activation of a haemolytic toxin.

DOI： 10.1038/ncomms7337

その他リンク： http://www.nature.com/articles/ncomms7337

記述言語：英語   掲載種別：研究論文（学術雑誌）  

Mutations of DJ-1 cause familial Parkin- son’s disease (PD), although the role of DJ-1 in PD remains unresolved. Very recent reports have shown that DJ-1 interacts with copper ions. This evidence opens new avenues to understanding the function of DJ-1 and its role in PD. Herein, we report that Zn(II) binds to DJ-1 with great selectivity among the other metals examined: Mn(II), Fe(II), Co(II), Ni(II), and Cu(II). High- resolution X-ray crystallography (1.18 Å resolution) shows Zn(II) is coordinated to the protein by the key residues Cys106 and Glu18. These results suggest that DJ- 1 may be regulated and/or stabilized by Zn(II).

DOI： 10.1021/bi500294h

その他リンク： http://pubs.acs.org/doi/abs/10.1021/bi500294h

記述言語：英語   掲載種別：研究論文（学術雑誌）  

Cell adhesion mediated by cadherins depends critically on the homophilic trans-dimerization of cadherin monomers from apposing cells, generating the so-called strand-swap dimer (ss-dimer). Recent evidence indicates that the ss-dimer is preceded by an intermediate species known as the X-dimer. Until now, the stabilized form of the X-dimer had only been observed in E-cadherin among the classical type I cadherins. Herein, we report the isolation and characterization of the analogous X-dimer of human P-cadherin. Small-angle X-ray scattering (SAXS) and site-directed mutagenesis data indicates that the overall architecture of the X-dimer of human P-cadherin is similar to that of E- cadherin. The X-dimerization is triggered by Ca2+ and governed by specific protein−protein interactions. The attachment of three molecules of Ca2+ with high affinity (Kd = 9 microM) stabilizes the monomeric conformation of P-cadherin (ΔTm = 17 °C). The Ca2+-stabilized monomer subsequently dimerizes in the X-configuration by establishing protein−protein interactions that require the first two extracellular domains of the cadherin. The homophilic X-dimerization is very specific, as the presence of the highly homologous E-cadherin does not interfere with the self-recognition of P-cadherin. These data suggest that the X-dimer could play a key role in the specific cell−cell adhesion mediated by human P-cadherin.

DOI： 10.1021/bi401341g

その他リンク： http://pubs.acs.org/doi/abs/10.1021/bi401341g

記述言語：英語   掲載種別：研究論文（学術雑誌）  

The optimization of antibodies is a desirable goal towards the development of better therapeutic strategies. The antibody 11K2 was previously developed as a therapeutic tool for inflammatory diseases, and displays very high affinity (4.6 pM) for its antigen the chemokine MCP-1 (monocyte chemo-attractant protein-1). We have employed a virtual library of mutations of 11K2 to identify antibody variants of potentially higher affinity, and to establish benchmarks in the engineering of a mature therapeutic antibody. The most promising candidates identified in the virtual screening were examined by surface plasmon resonance to validate the computational predictions, and to characterize their binding affinity and key thermodynamic properties in detail. Only mutations in the light-chain of the antibody are effective at enhancing its affinity for the antigen in vitro, suggesting that the interaction surface of the heavy-chain (dominated by the hot-spot residue Phe101) is not amenable to optimization. The single-mutation with the highest affinity is L-N31R (4.6-fold higher affinity than wild-type antibody). Importantly, all the single-mutations showing increase affinity incorporate a charged residue (Arg, Asp, or Glu). The characterization of the relevant thermodynamic parameters clarifies the energetic mechanism. Essentially, the formation of new electrostatic interactions early in the binding reaction coordinate (transition state or earlier) benefits the durability of the antibody-antigen complex. The combination of in silico calculations and thermodynamic analysis is an effective strategy to improve the affinity of a matured therapeutic antibody.

DOI： 10.1371/journal.pone.0087099

その他リンク： http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0087099

記述言語：英語   掲載種別：研究論文（学術雑誌）  

Near transporter (NEAT) domains of the iron-regulated surface determinant (Isd) proteins are essential for the import of nutritional heme from host animals to Gram-positive pathogens such as Staphylococcus aureus. The order of transfer of heme between NEAT domains occurs from IsdH to IsdA to IsdC, without any energy input despite the similarity of their three-dimensional structures. We measured the free energy of binding of heme and various metalloporphyrins to each NEAT domain and found that the affinity of heme and non-iron porphyrins for NEAT domains increased gradually in the same order as that for heme transfer. To gain insight into the atomistic mechanism for the differential affinities, we performed in silico molecular dynamics simulation and in vitro site-directed mutagenesis. The simulations revealed that the negatively charged residues that are abundant in the loop between strand β1b and the 3-10 helix of IsdH-NEAT3 destabilize the interaction with the propionate group of heme. The higher affinity of IsdC was in part attributed to the formation of a salt bridge between its unique residue, Glu88, and the conserved Arg100 upon binding to heme. In addition, we found that Phe130 of IsdC makes the beta7−beta8 hairpin less flexible in the ligand-free form, which serves to reduce the magnitude of the entropy loss on binding to heme. We confirmed that substitution of these key residues of IsdC decreased its affinity for heme. Furthermore, IsdC mutants, whose affinities for heme were lower than those of IsdA, transferred heme back to IsdA. Thus, NEAT domains have evolved the characteristic residues on the common structural scaffold such that they exhibit different affinities for heme, thus promoting the efficient transfer of heme.

DOI： 10.1021/bi4008325

その他リンク： http://pubs.acs.org/doi/abs/10.1021/bi4008325

記述言語：英語   掲載種別：研究論文（学術雑誌）  

CapE is an essential enzyme for the synthesis of capsular polysaccharide (CP) of pathogenic strains of Staphylococcus aureus. Herein we demonstrate that CapE is a 5-inverting 4,6-dehydratase enzyme. However, in the absence of downstream enzymes, CapE catalyzes an additional reaction (5-back-epimerization) affording a by-product under thermodynamic control. Single-crystal X-ray crystallography was employed to identify the structure of the by-product. The structural analysis reveals a network of coordinated motions away from the active site governing the enzymatic activity of CapE. A second dynamic element (the latch) regulates the enzymatic chemoselectivity. The validity of these mechanisms was evaluated by site-directed mutagenesis.

DOI： 10.1016/j.febslet.2013.10.009

その他リンク： http://www.sciencedirect.com/science/article/pii/S0014579313007667

記述言語：英語   掲載種別：研究論文（学術雑誌）  

The Isd (iron-regulated surface determinant) system of the human pathogen Staphylococcus aureus is responsible for the acquisition of heme from the host organism. We recently reported that the extracellular heme receptor IsdH-NEAT3 captures and transfers noniron antimicrobial porphyrins containing metals in oxidation state (III). However, it is unclear if geometric factors such as the size of the metal (ionic radius) affect binding and transfer of metalloporphyrins. We carried out an ample structural, functional, and thermodynamic analysis of the binding properties of antimicrobial indium(III)- porphyrin, which bears a much larger metal ion than the iron(III) of the natural ligand heme. The results demonstrate that the NEAT3 receptor recognizes the In(III)-containing PPIX in a manner very similar to that of heme. Site-directed mutagenesis identifies Tyr642 as the central element in the recognition mechanism as suggested from the crystal structures. Importantly, the NEAT3 receptor possesses the remarkable ability to capture dimers of metalloporphyrin. Molecular dynamics simulations reveal that IsdH-NEAT3 does not require conformational changes, or large rearrangements of the residues within its binding site, to accommodate the much larger (heme)2 ligand. We discuss the implications of these findings for the design of potent inhibitors against this family of key receptors of S. aureus.

DOI： 10.1002/pro.2276

その他リンク： http://onlinelibrary.wiley.com/doi/10.1002/pro.2276/abstract

記述言語：英語   掲載種別：研究論文（学術雑誌）  

Hydrogen bond networks are key elements of protein structure and function but have been challenging to study within the complex protein environment. We have carried out in-depth interrogations of the proton transfer equilibrium within a hydrogen bond network formed to bound phenols in the active site of ketosteroid isomerase. We systematically varied the proton affinity of the phenol using differing electron-withdrawing substituents and incorporated site-specific NMR and IR probes to quantitatively map the proton and charge rearrangements within the network that accompany incremental increases in phenol proton affinity. The observed ionization changes were accurately described by a simple equilibrium proton transfer model that strongly suggests the intrinsic proton affinity of one of the Tyr residues in the network, Tyr16, does not remain constant but rather systematically increases due to weakening of the phenol–Tyr16 anion hydrogen bond with increasing phenol proton affinity. Using vibrational Stark spectroscopy, we quantified the electrostatic field changes within the surrounding active site that accompany these rearrangements within the network. We were able to model these changes accurately using continuum electrostatic calculations, suggesting a high degree of conformational restriction within the protein matrix. Our study affords direct insight into the physical and energetic properties of a hydrogen bond network within a protein interior and provides an example of a highly controlled system with minimal conformational rearrangements in which the observed physical changes can be accurately modeled by theoretical calculations.

DOI： 10.1073/pnas.1302191110

その他リンク： http://www.pnas.org/content/110/28/E2552.long

記述言語：英語   掲載種別：研究論文（学術雑誌）  

Fragment-based drug discovery (FBDD) has enjoyed increasing popularity in recent years. We introduce SITE (single-injection thermal extinction), a novel thermodynamic methodology that selects high-quality hits early in FBDD. SITE is a fast calorimetric competitive assay suitable for automation that captures the essence of isothermal titration calorimetry but using significantly fewer resources. We describe the principles of SITE and identify a novel family of fragment inhibitors of the enzyme ketosteroid isomerase displaying high values of enthalpic efficiency.

DOI： 10.1021/jm301603n

その他リンク： http://pubs.acs.org/doi/abs/10.1021/jm301603n

記述言語：英語   掲載種別：研究論文（学術雑誌）  

CP (capsular polysaccharide) is an important virulence factor during infections by the bacterium Staphylococcus aureus. The enzyme CapF is an attractive therapeutic candidate belonging to the biosynthetic route of CP of pathogenic strains of S. aureus. In the present study, we report two independent crystal structures of CapF in an open form of the apoenzyme. CapF is a homodimer displaying a characteristic dumb-bell-shaped architecture composed of two domains. The N-terminal domain (residues 1–252) adopts a Rossmann fold belonging to the short-chain dehydrogenase/reductase family of proteins. The C- terminal domain (residues 252–369) displays a standard cupin fold with a Zn2 + ion bound deep in the binding pocket of the beta-barrel. Functional and thermodynamic analyses indicated that each domain catalyses separate enzymatic reactions. The cupin domain is necessary for the C3-epimerization of UDP-4-hexulose Meanwhile, the N-terminal domain catalyses the NADPH- dependent reduction of the intermediate species generated by the cupin domain. Analysis by ITC (isothermal titration calorimetry) revealed a fascinating thermodynamic switch governing the attachment and release of the coenzyme NADPH during each catalytic cycle. These observations suggested that the binding of coenzyme to CapF facilitates a disorder-to-order transition in the catalytic loop of the reductase (N-terminal) domain. We anticipate that the present study will improve the general understanding of the synthesis of CP in S. aureus and will aid in the design of new therapeutic agents against this pathogenic bacterium.

DOI： 10.1042/BJ20112049

その他リンク： http://www.biochemj.org/content/443/3/671.long

記述言語：英語   掲載種別：研究論文（学術雑誌）  

Iron is an essential nutrient for the proliferation of Staphylo- coccus aureus during bacterial infections. The iron-regulated surface determinant (Isd) system of S. aureus transports and metabolizes iron porphyrin (heme) captured from the host orga-. Transportation of heme across the thick cell wall of this bacterium requires multiple relay points. The mechanism by which heme is physically transferred between Isd transporters is largely unknown because of the transient nature of the interactions involved. Herein, we show that the IsdC transporter not only passes heme ligand to another class of Isd transporter, as previously known, but can also perform self-transfer reactions. IsdA shows a similar ability. A genetically encoded photoreactive probe was used to survey the regions of IsdC involved in self-dimerization. We propose an updated model that explicitly considers self-transfer reactions to explain heme delivery across the cell wall. An analogous photo-cross-linking strategy was employed to map transient interactions between IsdC and IsdE transporters. These experiments identified a key structural element involved in the rapid and specific transfer of heme from IsdC to IsdE. The resulting structural model was validated with a chimeric version of the homologous transporter IsdA. Overall, our results show that the ultra-weak interactions between Isd transporters are governed by bona fide protein structural motifs.

DOI： 10.1074/jbc.M112.346700

その他リンク： http://www.jbc.org/content/287/20/16477.long

記述言語：英語   掲載種別：研究論文（学術雑誌）  

Human P-cadherin is a promising therapeutic target against cancer. However, its characterization at the molecular level is still lacking. We report that human P-cadherin associated irreversibly in a distinct dimer configuration. Unexpectedly, the divalent cation Ca2+ was not necessary for dimerization, although it greatly stabilized the protein–protein complex.

DOI： 10.1039/c2mb25161b

その他リンク： http://pubs.rsc.org/en/content/articlelanding/2012/mb/c2mb25161b/unauth#!divAbstract

記述言語：英語   掲載種別：研究論文（学術雑誌）  

Enzymes synthesizing the bacterial CP (capsular polysaccharide) are attractive antimicrobial targets. However, we lack critical information about the structure and mechanism of many of them. In an effort to reduce that gap, we have determined three different crystal structures of the enzyme CapE of the human pathogen Staphylococcus aureus. The structure reveals that CapE is a member of the SDR (short-chain dehydrogenase/reductase) super-family of proteins. CapE assembles in a hexameric complex stabilized by three major contact surfaces between protein subunits. Turnover of substrate and/or coenzyme induces major conformational changes at the contact interface between protein subunits, and a displacement of the substrate-binding domain with respect to the Rossmann domain. A novel dynamic element that we called the latch is essential for remodelling of the protein–protein interface. Structural and primary sequence alignment identifies a group of SDR proteins involved in polysaccharide synthesis that share the two salient features of CapE: the mobile loop (latch) and a distinctive catalytic site (MxxxK). The relevance of these structural elements was evaluated by site-directed mutagenesis.

DOI： 10.1042/BSR20130017

その他リンク： http://www.bioscirep.org/content/33/3/e00043

記述言語：英語   掲載種別：研究論文（学術雑誌）  

ATP-binding cassette (ABC) transporters couple hydrolysis of ATP with vectorial transport across the cell membrane. We have reconstituted ABC transporter MsbA in nanodiscs of various sizes and lipid compositions to test whether ATPase activity is modulated by the properties of the bilayer. ATP hydrolysis rates, Michaelis–Menten parameters, and dissociation constants of substrate analog ATP-gamma-S demonstrated that physicochemical properties of the bilayer modulated binding and ATP- ase activity. This is remarkable when considering that the catalytic unit is located ~50 Å from the transmembrane region. Our results validated the use of nanodiscs as an effective tool to reconstitute MsbA in an active catalytic state, and highlighted the close relationship between otherwise distant transmembrane and ATPase modules.

DOI： 10.1016/j.febslet.2011.10.015

その他リンク： http://onlinelibrary.wiley.com/doi/10.1016/j.febslet.2011.10.015/abstract

記述言語：英語   掲載種別：研究論文（学術雑誌）  

Antibiotic resistance is increasingly seen as a serious problem that threatens public health and erodes our capacity to effectively combat disease. So-called non-iron metalloporhyrins have shown promising antibacterial properties against a number of pathogenic bacteria including Staphylococcus aureus. However, little is known about the molecular mechanism(s) of action of these compounds and in particular how they reach the interior of the bacterial cells. A popular hypothesis indicates that non-iron metalloporphyrins infiltrate into bacterial cells like a “Trojan horse” using heme transport systems. Iron-regulated surface determinant (Isd) is the best characterized heme transport system of S. aureus. Herein we studied the molecular mechanism by which the
extracellular heme-receptor IsdH-NEAT3 of Isd recognizes antimicrobial metalloporphyrins. We found that potent antibacterial porphyrins Ga(III)-protoporphyrin IX (PPIX) and Mn(III)- PPIX closely mimicked the properties of the natural ligand heme, namely (i) stable binding to IsdH-NEAT3 with comparable affinities for the receptor, (ii) nearly undistinghuishable three-dimensional structure when complexed with IsdH-NEAT3, and (iii) similar transfer properties to a second receptor IsdA. On the contrary, weaker antibacterial porphyrins Mg(II)-PPIX, Zn(II)- PPIX, and Cu(II)-PPIX were not captured effectively by IsdH-NEAT3 under our experimental conditions and displayed lower affinities. Moreover, reduction of Fe(III)-PPIX to Fe(II)-PPIX with dithionite abrogated stable binding to receptor. These data revealed a clear connection between oxidation state of metal and effective attachment to IsdH-NEAT3. Also, the strong correlation between binding affinity and reported antimicrobial potency suggested that the Isd system may be used by these antibacterial compounds to gain access to the interior of the cells. We hope these results will increase our understanding of Isd system of S. aureus and highlight its biomedical potential to deliver new and more efficient antibacterial treatments.

DOI： 10.1021/bi200493h

その他リンク： http://pubs.acs.org/doi/abs/10.1021/bi200493h

記述言語：英語   掲載種別：研究論文（学術雑誌）  

Human interleukin-11 (hIL-11) is a pleiotropic cytokine administered to patients with low platelet counts. From a structural point of view hIL-11 belongs to the long-helix cytokine superfamily, which is characterized by a conserved core motif consisting of four alpha-helices. We have investigated the region of hIL-11 that does not belong to the alpha-helical bundle motif, and that for the purpose of brevity we have termed “non-core region.” The primary sequence of the interleukin was altered at various locations within the non-core region by introducing glycosylation sites. Functional consequences of these modifications were examined in cell-based as well as biophysical assays. Overall, the data indicated that the non-core region modulates the function of hIL-11 in two ways. First, the majority of muteins displayed enhanced cell-stimulatory properties (superagonist behavior) in a glycosylation-dependent manner, suggesting that the non-core region is biologically designed to limit the full potential of hIL-11. Second, specific modification of a predicted mini alpha-helix led to cytokine inactivation, demonstrating that this putative structural element belongs to site III engaging a second copy of cell-receptor gp130. These findings have unveiled new and unexpected elements modulating the biological activity of hIL-11, which may be exploited to develop more versatile medications based on this important cytokine.

DOI： 10.1074/jbc.M110.152561

その他リンク： http://www.jbc.org/content/286/10/8085.long

記述言語：英語   掲載種別：研究論文（学術雑誌）  

N-Acylamino acids are a new family of versatile biological surfactants capable of extracting integral membrane proteins of various topologies from the biological membrane, in many instances surpassing the efficiency of commercial detergents.

DOI： 10.1039/b925791h

その他リンク： http://pubs.rsc.org/en/Content/ArticleLanding/2010/MB/b925791h#!divAbstract

記述言語：英語   掲載種別：研究論文（学術雑誌）  

The identification of hot spots, i.e., binding regions that contribute substantially to the free energy of ligand binding, is a critical step for structure-based drug design. Here we present the application of two fragment- based methods to the detection of hot spots for DJ-1 and glucocerebrosidase (GCase), targets for the development of therapeutics for Parkinson’s and Gaucher’s diseases, respectively. While the structures of these two proteins are known, binding information is lacking. In this study we employ the experimental multiple solvent crystal structures (MSCS) method and computational fragment mapping (FTMap) to identify regions suitable for the development of pharmacological chaperones for DJ-1 and GCase. Comparison of data derived via MSCS and FTMap also shows that FTMap, a computational method for the iden- tification of fragment binding hot spots, is an accurate and robust alternative to the performance of expensive and difficult crystallographic experiments.

DOI： 10.1007/s10822-009-9283-2

その他リンク： http://link.springer.com/article/10.1007/s10822-009-9283-2

記述言語：英語   掲載種別：研究論文（学術雑誌）  

Hydrogen bond networks are key elements of biological structure and function. Nevertheless, their structural properties are challenging to assess within complex macromolecules. Hydrogen-bonded protons are not observed in the vast majority of protein X-ray structures, and static crystallographic models provide limited information regarding the dynamical coupling within hydrogen bond networks. We have brought together 1.1-1.3 A ̊ resolution X-ray crystallography, 1H NMR, site-directed mutagenesis, and deuterium isotope effects on the geometry and chemical shifts of hydrogen-bonded protons to probe the conformational coupling of hydrogen bonds donated by Y16 and D103 in the oxyanion hole of bacterial ketosteroid isomerase. Our results suggest a robust physical coupling of the equilibrium structures of these two hydrogen bonds such that a lengthening of one hydrogen bond by as little as 0.01 A ̊ results in a shortening of the neighbor by a similar magnitude. Furthermore, the structural rearrangements detected by NMR in response to mutations within the active site hydrogen bond network can be explained on the basis of the observed coupling. The results herein elucidate fundamental structural properties of hydrogen bonds within the idiosyncratic environment of an enzyme active site and provide a foundation for future experimental and computational explorations of the role of coupled motions within hydrogen bond networks.

DOI： 10.1021/bi900713j

その他リンク： http://pubs.acs.org/doi/abs/10.1021/bi900713j

記述言語：英語   掲載種別：研究論文（学術雑誌）  

Human interleukin 15 (hIL15) is a soluble cytokine that plays a key role in the maintenance of long-lasting responses against pathogens and a valuable target for the treatment of autoimmune diseases. In this study, we sought to elucidate the thermodynamic basis of the recognition mechanism for its private α-receptor (hIL15Rα), considered the first step of the interleukin's activation pathway. Binding of wild-type hIL15 to its α-receptor is characterized by a very slow dissociation rate constant and driven by a favorable enthalpy change. We further studied the kinetic and energetic consequences of substituting residues of hIL15 located at the contact interface by means of the surface plasmon resonance technique. Replace- ment of negatively charged residues with Ala indicates that the energetics of interaction is primarily driven by electrostatic forces, manifested by a dramatic acceleration of the dissociation step and a reduction of favorable binding enthalpy. Our analyses also unveiled a novel and critical role for residue Tyr26 in the interaction, which facilitates desolvation of key charged residues during the assembly of the complex. These results were rationalized in terms of a previously reported structure of hIL15·hIL15α, demonstrating that the binding energetics is dominated by interactions occurring at three hot spots whose spatial locations coincide with a previously proposed structural division of the contact interface in three regions. Specifically, Region 1 is the main contributor to the binding energy of the complex by establishing very favorable electrostatic interactions with the receptor; Region 2 is also dominated by electrostatic forces, although of a lesser intensity; and Region 3 confers specificity to the association by means of high shape complementarity and by bringing additional stabilization energy to the complex. The biological impact of hIL15 mutations with the most effect on α-receptor binding was evaluated in a cell-based proliferation assay, validating the conclusions of our thermodynamic analyses and highlighting the functional importance of molecular contacts that promote prolonged binding of the interleukin to the α-receptor. In closing, the thermodynamics and physicochemical nature of the interactions observed in IL15h·IL15Rα complex, together with interactions in Region 3 of the interleukin, poses a stark contrast with the structurally related and sometimes functionally redundant interleukin 2.

DOI： 10.1016/j.jmb.2009.04.050

その他リンク： http://www.sciencedirect.com/science/article/pii/S0022283609005063

記述言語：英語   掲載種別：研究論文（学術雑誌）  

Enzymes are classically proposed to accelerate reactions by binding substrates within active- site environments that are structurally preorganized to optimize binding interactions with reaction transition states rather than ground states. This is a remarkably formidable task considering the limited 0.1-1 Å scale of most substrate rearrangements. The flexibility of active-site functional groups along the coordinate of substrate rearrangement, the distance scale on which enzymes can distinguish structural rearrangement, and the energetic significance of discrimination on that scale remain open questions that are fundamental to a basic physical understanding of enzyme active sites and catalysis. We bring together 1.2-1.5 Å resolution X-ray crystallography, 1H and 19F NMR spectroscopy, quantum mechanical calculations, and transition-state analogue binding measurements to test the distance scale on which noncovalent forces can constrain the structural relaxation or translation of side chains and ligands along a specific coordinate and the energetic consequences of such geometric constraints within the active site of bacterial ketosteroid isomerase (KSI). Our results strongly suggest that packing and binding interactions within the KSI active site can constrain local side-chain reorientation and prevent hydrogen bond shortening by 0.1 Å or less. Further, this constraint has substantial energetic effects on ligand binding and stabilization of negative charge within the oxyanion hole. These results provide evidence that subtle geometric effects, indistinguish- able in most X-ray crystallographic structures, can have significant energetic consequences and highlight the importance of using synergistic experimental approaches to dissect enzyme function.

DOI： 10.1021/ja803928m

その他リンク： http://pubs.acs.org/doi/abs/10.1021/ja803928m

記述言語：英語   掲載種別：研究論文（学術雑誌）  

Equinatoxin-II is a eukaryotic pore-forming toxin be- longing to the family of actinoporins. Its interaction with model membranes is largely modulated by the presence of sphingomyelin. We have used large unilamellar vesicles and lipid monolayers to gain further information about this interaction. The coexistence of gel and liquid-crystal lipid phases in sphingomyelin/phosphatidylcholine mixtures and the coexistence of liquid-ordered and liquid-disordered lipid phases in phosphatidylcholine/cholesterol or sphingomyelin/phosphatidylcholine/cholesterol mixtures favor membrane insertion of equinatoxin-II. Phosphatidylcholine vesicles are not permeabilized by equinatoxin-II. However, the localized accumulation of phospholipase C-generated diacylglycerol creates conditions for toxin activity. By using epifluorescence microscopy of transferred monolayers, it seems that lipid packing defects arising at the interfaces between coexisting lipid phases may function as preferential binding sites for the toxin. The possible implications of such a mechanism in the assembly of a toroidal pore are discussed.

DOI： 10.1074/jbc.M313817200

その他リンク： http://www.jbc.org/content/279/33/34209.long

記述言語：英語   掲載種別：研究論文（学術雑誌）  

Lyophilized horseradish peroxidase (HRP) ex- hibits poor stereoselectivity in the sulfoxidation of thio- anisole when the enzyme is either redissolved in water or suspended in organic solvents. However, when HRP is co-lyophilized in the presence of lyoprotectants or ligands, its stereoselectivity, although still low in most organic solvents, increases up to 4-fold if assayed in secondary or tertiary alcohols (but not in their linear isomers).Amechanistichypothesisispresentedex- plaining this puzzling phenomenon on the basis of a model of the active site of the enzyme-substrate complex derived from its X-ray crystal structure by means of molecular dynamics and energy minimization.

DOI： 10.1002/bit.10308

その他リンク： http://onlinelibrary.wiley.com/doi/10.1002/bit.10308/full

記述言語：英語   掲載種別：研究論文（学術雑誌）  

The oxidation of aryl alkyl sulfides with H2O2 in aqueous solution is a reasonably facile reaction produc- ing racemic sulfoxides. We show that in the presence of the hydrolytic enzyme a-chymotrypsin such a sulfoxida- tion is accelerated and, more importantly, becomes stereoselective. With phenyl isobutyl sulfide as a model, the chymotrypsin-mediated, highly asymmetric oxida- tion is shown to occur in the hydrophobic binding pocket of the enzyme active site. The stereoselectivity of the chymotrypsin-mediated sulfoxidations is correctly ex- plained by means of structure-based molecular modeling of the enzyme-sulfide complexes.

DOI： 10.1002/bit.10187

その他リンク： http://onlinelibrary.wiley.com/doi/10.1002/bit.10187/full

記述言語：英語   掲載種別：研究論文（学術雑誌）  

Horseradish peroxidase exhibits a meager stereoselectivity (E) in the sulfoxidation of thioanisole (1a) in 99.8% (v/v) methanol. The E value, however, is greatly enhanced when the enzyme forms a complex with benzohydroxamic acid (2a). These findings are rationalized by means of molecular dynamics simulations and energy minimization which correctly explain (i) why the free enzyme is not stereoselective, (ii) why 2a inhibits peroxidase-catalyzed sulfoxidation of 1a but the enzymatic formation of one enantiomer of the sulfoxide product is inhibited much more than that of the other, thereby raising peroxidase’s E, and (iii) why in the presence of 2a the enzyme favors production of the S sulfoxide of 1a. The generality of the observed ligand-induced stereoselectivity enhancement is demonstrated with other hydrophobic hydroxamic acids, as well as with additional thioether substrates.

DOI： 10.1021/ja012075o

その他リンク： http://pubs.acs.org/doi/abs/10.1021/ja012075o

記述言語：英語   掲載種別：研究論文（学術雑誌）  

Equinatoxin II is a 179-amino-acid pore-forming protein isolated from the venom of the sea anemone Actinia equina. Large unilamellar vesicles and lipid monolayers of different lipid compositions have been used to study its interaction with membranes. The critical pressure for insertion is the same in monolayers made of phosphatidylcholine or sphingomyelin (~26 mN m-1) and explains why the permeabilization of large unilamellar vesicles by equinatoxin II with these lipid compositions is null or moderate. In phosphatidylcholine-sphingomyelin (1:1) monolayers, the critical pressure is higher (~33 mN m-1), thus permitting the insertion of equinatoxin II in large unilamellar vesicles, a process that is accompanied by major conformational changes. In the presence of vesicles made of phosphatidylcholine, a fraction of the protein molecules remains associated with the membranes. This interaction is fully reversible, does not involve major conformational changes, and is governed by the high affinity for membrane interfaces of the protein region comprising amino acids 101–120. We conclude that although the presence of sphingomyelin within the membrane creates conditions for irreversible insertion and pore formation, this lipid is not essential for the initial partitioning event, and its role as a specific receptor for the toxin is not so clear-cut.

その他リンク： http://www.sciencedirect.com/science/article/pii/S0006349501761073

記述言語：英語   掲載種別：研究論文（学術雑誌）  

To study the activity and mechanism of pore formation of equinatoxin-II, a cytolysin isolated from a sea anemone, we use different membrane models i.e. cell membranes, liposomes and lipid monolayers. In this work we have determined to what extent the results obtained in one system can be extrapolated to the others.

その他リンク： http://bijh-s.zrc-sazu.si/abs/SI/ABS/index.asp

記述言語：英語   掲載種別：研究論文（学術雑誌）  

The interaction of the wheat antibacterial peptide a-thionin with large unilamellar vesicles has been investigated by means of fluorescence spectroscopy. Binding of the peptide to the vesicles is followed by the release of vesicle contents, vesicle aggregation, and lipid mixing. Vesicle fusion, i.e., mixing of the aqueous contents, was not observed. Peptide binding is governed by electrostatic interactions and shows no cooperativity. The amphipatic nature of wheat a-thionin seems to destabilize the membrane bilayer and trigger the aggregation of the vesicles and lipid mixing. The presence of distearoylphosphatidylethanolamine-poly(ethy1eneglycol 2000) (PEG-PE) within the membrane provides a steric barrier that inhibits vesicle aggregation and lipid mixing but does not prevent leakage. Vesicle leakage through discrete membrane channels is unlikely, because the release of encapsulated large fluorescent dextrans is very similar to that of 8-arninonaphthalene-l,3,6,trisulfonic acid (ANTS). A minimum number of 700 peptide molecules must bind to each vesicle to produce complete leakage, which suggests a mechanism in which the overall destabilization of the membrane is due to the formation of transient pores rather than discrete channels.

その他リンク： http://onlinelibrary.wiley.com/doi/10.1002/pro.5560071210/abstract

記述言語：英語   掲載種別：研究論文（学術雑誌）  

The effects of five antipathogenic plant peptides, wheat alpha-thionin, potato PTH1 defensin, barley LTP2 lipid transfer protein, and potato tuber DL1 and DL2 defensins, have been tested against phospholipid vesicles (liposomes). Wheat thionin very actively induces aggregation and leakage of negatively charged vesicles. LTP2 displays the same activities, although to a limited extent. Under certain conditions PTH1 and DL2 induce vesicle aggregation, but not leakage. Potato defensin DL1 failed to show any effect on liposomes. The same peptides have been assayed against a plant pathogenic bacterium, both the membrane-active and -inactive compounds having efficient antibacterial action.

その他リンク： http://www.sciencedirect.com/science/article/pii/S0014579397006133

記述言語：英語   掲載種別：研究論文（学術雑誌）  

A variety of structural techniques, including IR spectroscopy, reveals that thermal denaturation of bacteriorhodopsin follows a given pathway (successively rearrangement of helical structures, extensive deuterium exchange, and finally protein aggregation) irrespective of heating rate, pH or ionic strength conditions. In all cases, thermal denaturation leads to a 'compact denatured state' which retains a large proportion of ordered structure.

その他リンク： http://onlinelibrary.wiley.com/doi/10.1016/0014-5793%2895%2900570-Y/full

記述言語：英語   掲載種別：研究論文（学術雑誌）  

The translational and rotational electrokinetics of the anesthetic-induced spectral transitions bR568 -> bR480 -> bR380 of bacteriorhodopsin have been investigated. Formation of the bR480 form is associated with an increase of the purple membrane negative electrokinetic charge, while the transformation of bR480 into bR380 is accompanied by a decrease of the membrane negative charge as compared to that of the 480 rim-absorbing form. Removal of anesthetics leads to the back transitions bR480 -> bR568 and (in part) bR380 ~ bR568; however, the electrokinetic charge of the native membranes is not restored. A strong decrease in the electric polarizability and the appearance of a slow polarizability component are also observed in anesthetic-treated membranes. Comparison with the electrokinetic behaviour of partially delipidated membranes and with that of liposomes composed of purple membrane total lipids suggests that: (i) anesthetic molecules partition mainly at the protein/lipid interface inducing irreversible rearrangement of the boundary lipid layer, and (ii) different mode(s) or site(s) of interaction are responsible for the spectral and surface charge effects. The data are compatible with the hypothesis of anesthetics acting through partial dehydration of the membrane surface.

その他リンク： http://www.sciencedirect.com/science/article/pii/000527369500074D

開催年月日： 2023年7月 - 2024年7月

記述言語：日本語   会議種別：口頭発表（一般）  

開催地：名古屋国際会議場   国名：日本国  

IOX40は腫瘍壊死因子受容体 (TNFR)ファミリーに属する蛋白質であり、OX40Lはそのリガンドとして機能する腫瘍壊死因子(TNF)である。OX40-OX40L間の蛋白質-蛋白質間相互作用(PPI)はNF-κシグナルを惹起することによるT細胞活性化に寄与しているが、これらの蛋白質の過剰発現は自己免疫疾患の原因となることも報告されている。そのため、OX40-OX40L間PPIは重要な創薬標的である。OX40-OX40Lの複合体結晶構造より、ホモ3量体を形成するOX40Lに対して3分子のOX40が相互作用する特徴的なPPIであることが報告されている。本研究では物理化学的手法によってそのようなPPIの精密解析を実施し、相互作用メカニズムの解明を目指した。 　本研究ではまずOX40及びOX40Lの膜外ドメインを哺乳類細胞発現系により発現・精製した。得られた組換え蛋白質を用い、表面プラズモン共鳴法(SPR)によりOX40-OX40L間PPIの測定系を確立した。この系を活用して相互作用に伴い溶媒露出度が著しく低下するOX40のアミノ酸残基を中心にアラニンスキャニングを実施すると、PPIが著しく損なわれるホットスポットと呼べる残基を特定することができた。さらに、アラニンスキャニングにおける相互作用の速度論パラメーターの変化に基づき、OX40-OX40L間相互作用プロセスを考察した。

開催年月日： 2023年7月 - 2024年7月

記述言語：日本語   会議種別：口頭発表（一般）  

開催地：名古屋国際会議場   国名：日本国  

Iヒト免疫不全ウイルス（HIV）に対抗する抗体の開発において問題となっているのが、取得される抗HIV抗体の主なエピトープがアミノ酸保存性の低いエンベロープスパイク（ENV）部位であることにある。そのため、すべてのHIVウイルスに対して高い中和活性をもつ抗体を取得することは難しい。まれに、ほとんどのHIVウイルス間でアミノ酸が保存されている膜近位領域（MPER）に対する抗体が得られ、広い中和活性をもちうるが、その活性は低い場合が多く、医薬品への応用には至っていない。 　一方、近年我々は、芳香環を有する低分子を抗MPER抗体に修飾することにより中和活性が著しく向上することを報告している。しかしながら、膜に埋め込まれたMPERの取り扱いが難しく、低分子付加MPER抗体とMPERの相互作用メカニズムは未だに解明されていない。本研究ではMPERと膜貫通領域（TMD）からなる疎水性が高く調製の難しいペプチドを取り扱う系を確立し、構造的および熱力学的な観点からの相互作用を評価することによって、結合に関与するアミノ酸の同定や、化学修飾基の膜環境中抗原認識への寄与について理解することを目指している。本発表では、MPERとTMDからなるペプチドの調製法と得られたペプチドを用いた抗体との相互作用の評価の結果について報告する。本研究の進展により、膜付近のウイルス抗原を標的とした抗体ベースの新規モダリティの開発の進展が期待される。

開催年月日： 2023年7月 - 2024年7月

記述言語：日本語   会議種別：口頭発表（一般）  

開催地：名古屋国際会議場   国名：日本国  

IgG分子はヒトの体内において主に体液性免疫に関わる重要な分子であり、創薬モダリティーとして近年研究開発が進められている。IgG分子は抗原を認識するFab領域、Fc受容体や補体系と相互作用することで免疫系を活性化するFc領域、それら二つの機能領域をつなぐヒンジ領域から構成される。IgG分子はヒンジ領域が持つ柔軟性の影響で構造解析が困難であり、分子全体としての作用機序は不明な点が多い。本研究ではヒンジ領域の役割を明らかにするために、IgG1のヒンジ領域の下部に注目して変異を導入し、IgG1と高い親和性で相互作用することが知られているFc受容体のFcγRⅠとの相互作用を評価した。本研究を通じて得られたヒンジ領域下部領域の1アミノ酸残基を欠失した変異体は、ヒンジ領域の二つのジスルフィド結合およびCH3ドメイン間の相互作用の両方が失われ、一対の重鎖と軽鎖を持つ、半分子の抗体となった。この抗体半分子は、野生型IgGと比較して親和性はやや低下していたものの、FcγRⅠとの相互作用を保っていた。また、HS-AFM計測より、この抗体半分子は1:1または1:2でFcγRⅠと相互作用することが明らかとなった。本研究より、ヒンジ領域の下部が抗体の高次構造形成に重要である一方、抗体半分子でもFcγRⅠとの相互作用が可能であることが明らかとなった

開催年月日： 2020年7月

記述言語：英語  

開催地：Hokkaido   国名：日本国  

Development of new antibiotics is urgently needed to combat emerging resistance in bacteria. MsbA is an essential ABC-transporter located in the inner membrane of E. coli. MsbA is directly responsible for the export of Lipid A, the key precursor of lipopolysaccharide (LPS, also known as endotoxin). LPS is of critical importance for the bacterium survival, and therefore MsbA is a pharmacological target. Because of continuous improvements in biophysical methodologies, these techniques are increasingly employed for the screening of chemical compounds to protein targets. In this study, we sought to perform such screening in MsbA by surface plasmon resonance (SPR), a methodology that detects molecules binding to the target with great celerity. To enable the screening, we embedded MsbA in proteo-lipidic particles known as nanodiscs, which greatly stabilized the ATPase activity of the target. Despite the large difference in mass between the dimeric MsbA in nanodiscs and the average size of fragments (>1,000-fold) we detected robust binding of several compounds. The binding of these compounds was cross-validated by dose-dependence analysis and thermodynamic techniques. Moreover, the majority of these fragment compounds did not only bind to the target molecule, but also reduced the ATPase activity of MsbA with various intensities, from partial to complete inhibition. Our research has revealed the identity of novel compounds inhibiting the activity of an essential bacterial protein. We hope to develop these fragments into potent antibacterial compounds, and take the lessons learned to apply this approach to other protein targets of bacterial and human origin.

その他リンク： https://www.proteinsociety.org/2020wcps

開催年月日： 2020年2月

記述言語：英語  

開催地：San Francisco   国名：アメリカ合衆国  

Staphylococcus aureus is a pathogenic bacterium responsible for a majority of deadly hospital-acquired infections worldwide, and increasingly resistant to a broad range of antibiotics. This dangerous combination demands action to obtain more efficient antimicrobial approaches. The iron surface determinant (Isd) system comprises a group of S. aureus proteins that obtain iron from the host organism. Since iron is essential for bacterial survival and proliferation during infection, Isd has been proposed as an antibacterial target. In particular, the protein IsdH is exposed on the surface of the bacteria and able to sequester hemoglobin to acquire its heme molecule which contains an atom of iron. IsdH comprises three near-iron transporter (NEAT) domains binding hemoglobin (NEAT1 and NEAT2) and heme (NEAT3) connected by characteristic linker domains of unclear function. The objective of this study is to deciphering the molecular mechanism of heme extraction, including the role of the linker re- gion. The first evidence of the structure of IsdH linker-NEAT3 at high resolu- tion with heme bound is presented. In combination with thermodynamic and structural data we propose a extraction mechanism of heme by IsdH. Although the linker neither accelerate nor strengthen the binding of heme, it contributes to stabilize the critical NEAT3 domain necessary to capture heme. This research contributes to clarify the molecular basis for heme extraction by a po- tential antimicrobial target.

その他リンク： https://www.biophysics.org/2020meeting#/

開催年月日： 2020年2月

記述言語：英語  

開催地：San Francisco   国名：アメリカ合衆国  

Broadly-neutralizing antibodies (bnAbs) against the Env glycoprotein guide the rational development of vaccines and therapeutic agents to prevent or treat HIV- 1 infection. In addition, bnAb-derived binding fragments are instrumental for the study of the structure-function relationships of the conformationally dynamic Env complex. Here, we demonstrate that the anti-viral activity of bnAbs against the Membrane-Proximal External Region of the Env glycoprotein can be enhanced via single-site modification of membrane-interacting Ab areas with synthetic aromatic compounds. Potency enhancement in cell-entry inhibition and standard neutralization assays correlated with an increase in affinity for the native antigen in virions and did not compromise neutralization breadth. Thus, we have established an opti- mization procedure with the potential of improving functionality of Abs that bind immunotherapeutic targets at membrane surfaces.

その他リンク： https://www.biophysics.org/2020meeting#/

開催年月日： 2019年12月

記述言語：英語  

開催地：San Diego   国名：アメリカ合衆国  

Engineered variants of broadly-neutralizing antibodies (bnAbs) have shown efficacy in passive immunotherapy of HIV-1 infection. Here, we demonstrate that the anti-viral activity of bnAbs against the Membrane-Proximal External Region of the HIV-1 Env glycoprotein can be enhanced up to 103-fold, via single-site modification of membrane-interacting areas with linear and polycyclic aromatic chemical compounds. In an extreme case, chemical modification of a distant site complements ablation of the heavy-chain complementarity determining region 3 (HCDR3) loop apex, resulting in the functional recovery of a fully inactive antibody. Thus, we have established a procedure based on the use of synthetic molecules to improve functionality of antibodies against HIV.

その他リンク： https://www.antibodysociety.org/2019-antibody-engineering-therapeutics/

開催年月日： 2019年11月

記述言語：英語  

開催地：Lisbon Congress Center, Lisbon   国名：ポルトガル共和国  

Engineered variants of broadly-neutralizing antibodies (bnAbs) have shown efficacy in passive immunotherapy of HIV-1 infection. Particularly, bnAbs raised against the neutralizing MPER epitope have gathered much attention in the area because they can potentially block infection by the large variety of circulating HIV-1 strains and isolates. Despite their remarkable breadth, the potential clinical translation of Abs belonging to this class is hampered by their overall modest potency. Here, we employ cell-entry inhibition and ex-vivo infection neutralization assays to demonstrate that the potency of the MPER bnAb 10E8, can be enhanced up to 102-fold, via single-site modification with linear and polycyclic aromatic chemical compounds. That is, chemically modified 10E8 versions display potency levels comparable to those of bnAbs currently under clinical development. Thus, we have established a procedure based on the use of synthetic molecules to improve functionality of pan-neutralizing HIV antibodies.

開催年月日： 2019年10月 - 2020年10月

記述言語：英語  

開催地：Tokyo   国名：日本国  

The bacterium Staphylococcus aureus is the major cause of deadly hospital-acquired infections worldwide1. The Isd (Iron surface determinant) system is a group of proteins that obtain iron from the host organism2,3, helping the bacterium to proliferate, and therefore a promising antibacterial target. The protein IsdH binds to hemoglobin and acquires the heme4,5. IsdH comprises three NEAT (Near-iron transporter) domains connected by linkers of unknown function6,7. The objectives of this study are deciphering the molecular mechanism of heme extraction, to explain the role of the linker region and to obtain an antibody that recognizes IsdH specifically while simultaneously inhibiting heme binding. The first evidence of the structure of IsdH linker-NEAT3 bound to heme was reported in this research. The heme extraction mechanism of IsdH has been deciphered and the role of the linker as key for heme extraction has been explained through structure analysis. Herein we also report a novel VHH that binds to IsdH linker-NEAT3. The crystal structure of the complex of VHH bound to -NEAT3 was determined at high-resolution, showing that the VHH specifically recognizes the heme binding pocket, and explaining why this antibody inhibits heme acquisition by IsdH.

その他リンク： https://cbi-society.org/taikai/taikai19/index_e.html

開催年月日： 2019年10月 - 2020年10月

記述言語：英語  

開催地：Tokyo   国名：日本国  

Many proteins have both rigid and flexible regions, and change their conformations upon ligand binding. With current methodologies of antibody generation, it is often difficult to rationally obtain specific antibodies toward flexible epitopes on antigens due to the lack of our knowledge on molecular recognition between antibodies and flexible antigens. In this study, we focused on the interaction between an antibody and an antigen binding to metal ions, to obtain quantitative relationships about antibody-antigen interactions where the antigen is highly flexible. As a model system, we used the Fab region of a monoclonal antibody 5E1 whose antigen is Sonic hedgehog (Shh) protein, which also binds to Ca2+ and Zn2+ ions. In a previous study, 5E1 Fab had exhibited high affinity toward Shh regardless of the presence or absence of the metal ions [1]. However, the details of the interactions and physicochemical properties remains unclear. In this study, to reveal the binding mechanism of 5E1 Fab and Shh, thermodynamic and kinetic parameters of the interactions were experimentally evaluated, and we observed drastic changes of binding enthalpy and entropy depending on the existence of the metal ions. In addition, we analyzed and discussed energetic contributions of some interface residues based on physicochemical measurements and molecular dynamics simulations with the Ala mutants as well as the wild-type. In addition to the interaction analyses, we also experimentally and computationally assessed the thermal stability of Shh with or without the metal ions, showing the dependency of the metal ions on the thermal stability of the antigen. A co-crystal structure of Shh and 5E1 Fab with the metal ions was already solved [1]. In this study, we also succeeded in obtaining a crystal structure of the complex without the metal ions, enabling the structure-based understanding of the interaction with or without the metal ions. Putting together, our results could be a guideline for rational design of antibodies that recognize flexible antigens.

その他リンク： https://cbi-society.org/taikai/taikai19/index_e.html

開催年月日： 2019年7月

記述言語：英語   会議種別：口頭発表（一般）  

開催地：Madrid   国名：スペイン  

Binding of antibodies (Abs) 4E10 or 10E8 to the envelope gly- coprotein (Env) of HIV-1 exerts one of the broadest neutralizing activities against this virus described to date. These Abs recognize the helical juncture that connects the membrane-proximal external region (MPER) and the transmembrane domain (TMD) present in the Env subunit gp41. Thus, gaining insight into the mechanism underlying MPER-mediated neutralization breadth can contribute to developing more effective vaccines and immunotherapeutic agents against HIV. Both, 4E10 and 10E8, exhibit unusual adaptations to attain specific, high-affinity binding to the helical MPER epitope presented on the surface of the viral membrane interface. We report here that manipulation through mutagenesis can adjust the 4E10 and 10E8 paratope surfaces to the viral membrane through semi-specific electrostatic interactions, and thereby modulate their biological activity.

その他リンク： https://www.sebbm.es/web/en/conferences/sebbm-conferences/3029-madrid-2019

開催年月日： 2019年6月 - 2019年7月

記述言語：英語  

開催地：Seattle   国名：日本国  

Since phosphorylation of an amino acid plays important roles in various cellular processes, how phos- phorylated amino acids are recognized has been widely discussed. However, many previous studies assumed that the protonation state of a phosphate group was (PO32) despite the fact that the phosphate group (pKa ~7) would exist as an equilibrium mixture of the unprotonated state (PO32-) and the singly protonated state (PO3H) in physiological condition. To analyze effects of a protonation state on protein dynamics, we performed MD simulations of 4 different proteinphosphorylated peptide complexes in vari- ous biological processes in which each peptide contained a phosphoserine residue in the unprotonated or singly protonated state. Our result showed that the (PO32-) was more preferable to (PO3H-) in the interactions due to the larger mobility of the phosphate group in the (PO32-) state. Furthermore, we also obtained a monoclonal antibody toward a phosphorylated peptide by phage display, and X-ray crystal- lography, thermodynamic analysis, mutagenesis, and MD simulations showed this antibody captures both protonation states equally well. Putting together, our results suggest that even a single protonation could have a large effect on molecular recognition of a phosphate group.

その他リンク： https://www.proteinsociety.org/e/in/eid=4

開催年月日： 2019年3月

記述言語：英語   会議種別：口頭発表（一般）  

開催地：福岡市休暇村志賀島   国名：日本国  

The remarkable breadth of broadly neutralizing antibodies (bNAbs) against the transmembrane protein gp41 of HIV-1 makes this type of antibodies suitable model system for structure-guided vaccine design and immunotherapeutics against AIDS. In particular, bNAbs directed against the membrane proximal external region (MPER) of gp41 are considered a paradigm because of the mechanism of recognition of the antigen seem to involve neighboring lipids of the viral membrane. Indeed, the concrete role of these lipids for the biological function of these bNAbs is still a subject of controversy. In this presentation I will address this debate for two bNAbs: 4E10 and 10E8. First, we compared the partitioning ability of the 4E10 antibody and several of its variants in synthetic membranes (liposomes). The antibody was mainly mutated at the region of the paratope surface in contact with the membrane interface. We employed a physical separation approach (vesicle flotation) and quantitative fluorescence measurements (spectroscopic titration) to evaluate the interaction with the membrane. Our energetic analysis suggested that antibody 4E10 behaves as a peripheral membrane protein. Second, we analyzed the structure and binding activities of the 10E8 antibody in membrane and membrane-like environments. The X-ray crystal structure of the Fab-peptide complex in detergents revealed for the first time that the epitope of 10E8 comprises a continuous helix spanning the gp41 MPER/transmembrane domain junction. Overall, these findings support the idea that lipids are critical for the binding mechanism of these antibodies. We hope this research will help to envision affordable and efficient vaccines against HIV-1.

開催年月日： 2018年11月 - 2019年11月

記述言語：英語   会議種別：口頭発表（一般）  

開催地：Kuala Lumpur   国名：マレーシア  

The discovery of novel medicines to tackle human diseases such as cancer or autoimmune disorders is a pressing challenge that requires urgent attention from the medical and scientific communities. The incorporation of proteins in the therapeutic arsenal is changing the perception of how medicines work. In particular, therapeutic antibodies and antibody-based molecules comprise a large fraction of the total substances approved by regulatory agencies to treat various diseases during the last decade. In this presentation, I will describe our efforts to understand the mechanisms by which antibodies recognize antigens. For example, the unprecedented role of membrane lipids in the recognition of the HIV-1 protein gp41 by broadly neutralizing antibodies of the IgG1 class will be described in this presentation. Another key element of antibody function is the cellular responses triggered by the binding of immune-complexes to Fc. Factors modulating the cytotoxic effect of antibodies such as glycosylation will be discussed in structural and energetic terms. Our research contributes to understand the fundamental basis of antibody-antigen and antibody-receptor interactions, directing ongoing efforts to design proteins with even better therapeutic outcomes.

開催年月日： 2018年9月

記述言語：英語   会議種別：公開講演，セミナー，チュートリアル，講習，講義等  

開催地：Department of Molecular Medicine, SickKids Research Institute, Toronto   国名：カナダ  

開催年月日： 2018年9月

記述言語：英語   会議種別：公開講演，セミナー，チュートリアル，講習，講義等  

開催地：National Center for Advancing Translational Sciences   国名：アメリカ合衆国  

開催年月日： 2018年9月

記述言語：英語   会議種別：シンポジウム・ワークショップ パネル（公募）  

開催地：National Center for Advancing Translational Sciences   国名：アメリカ合衆国  

その他リンク： https://ncats.nih.gov/expertise/preclinical/agm/training/sept2018

開催年月日： 2018年6月 - 2018年7月

記述言語：英語   会議種別：公開講演，セミナー，チュートリアル，講習，講義等  

開催地：Sant Feliu de Guixols, Girona   国名：スペイン  

The binding of antibodies of the IgG class to Fcgamma receptors triggers cell effector functions critical for the successful immune response. At the molecular level, the recognition of the Fc region of IgG involves an intricate network of non-covalent and weak interactions leading a broad range of affinities. As in drug binding to its biological target, or an inhibitor to an enzyme, changes in this carefully crafted three-dimensional arrangement of atoms leads to profound differences in the strength of protein-receptor partnerships. The small differences in the sequence of Fcgamma receptors lead to a broad range of binding responses with biological consequences. In this presentation the molecular mechanisms explaining the unique strength of the binding between IgG1-Fc and FcgammaRI (high-affinity receptor) will be analyzed from structural and thermodynamic points of view, including a discussion in depth about two antagonistic mechanisms proposed in recent times. In addition to geometric factors, it is increasingly evident that dynamic aspects of the binding partners also influence the interaction between IgG and Fcgamma receptors. In particular, the role of the glycan portion attached to both antibody and receptor is essential for the intensity of the biological response. By engineering a Fcgamma receptor, the role of the glycans is further clarified, suggesting that the composition of the glycan influence the degree of mobility (disorder) of the Fc portion of IgG. Overall, the accumulating evidence suggest that multifaceted interactions between IgG-Fc and Fcgamma receptors explain, at least in part, the immune effector function.

その他リンク： http://meetings.embo.org/event/18-antibodies

開催年月日： 2018年2月

記述言語：英語  

開催地：San Francisco   国名：アメリカ合衆国  

Binding of antibodies 4E10 or 10E8 to the helical juncture that connects the membrane-proximal external region (MPER) and the transmembrane domain (TMD) of the Envelope glycoprotein (Env) gp41 subunit, translates into one of the broadest neutralizing activities against HIV-1 described to date. Accordingly, these antibodies and their mechanisms of action provide timely models in the development of effective vaccination and immunotherapeutic strategies. Both, 4E10 and 10E8, exhibit unusual adaptations to attain specific, high-affinity binding to the helical MPER epitope anchored into the viral membrane interface. We report here that manipulation of the paratope surface through mutagenesis can be used to adjust the electrostatic interactions of 4E10 and 10E8 with the membrane, and modulate their biological activity (neutralization potency and polyreactivity).

その他リンク： https://www.biophysics.org/past-annual-meetings/62nd-annual-meeting

開催年月日： 2018年2月

記述言語：英語   会議種別：公開講演，セミナー，チュートリアル，講習，講義等  

開催地：Institute Juan March, Madrid   国名：スペイン  

開催年月日： 2017年12月

記述言語：英語   会議種別：公開講演，セミナー，チュートリアル，講習，講義等  

開催地：Faculty of Science and Technology, The University of the Basque Country, Bilbao   国名：スペイン  

Dr. Caaveiro’s research program combines structural and thermodynamic data to address a broad range of biologically inspired phenomena. His ultimately goal is to understand the physicochemical basis underpinning biomolecular recognition at the atomic scale. Recently, Dr. Caaveiro and co-workers has revealed an unprecedented role for lipids in the pore-forming mechanism of the potent hemolytic class of actinoporins.

開催年月日： 2017年9月

記述言語：英語   会議種別：公開講演，セミナー，チュートリアル，講習，講義等  

開催地：Kumamoto University, Kumamoto   国名：日本国  

Pore-forming toxins (PFTs) are metamorphic proteins that fold stably in water solutions as well as in cellular membranes, where they assemble to form lethal transmembrane holes producing cell death. Actinoporins are a family of potent hemolytic toxins vigorously studied as a paradigm of alpha-helical PFTs in the context of lipid–protein interactions. We have revealed for the first time the complete energetic and structural basis of the transformation of a PFT from the water-soluble to the active transmembrane state, and its reverse process. We will show that FraC, an actinoporin, undergoes a stepwise metamorphosis towards the lethal pore that is catalyzed by the physicochemical properties of the membrane and by key non-covalent interactions with specific lipids.

その他リンク： https://www.aeplan.co.jp/bsj2017/

開催年月日： 2017年6月 - 2019年6月

記述言語：英語   会議種別：口頭発表（一般）  

開催地：Cadiz   国名：スペイン  

Structural basis of HIV entry and its inhibition by neutralizing antibodies against the fusogenic glycoprotein subunit 41

開催年月日： 2017年3月

記述言語：英語   会議種別：公開講演，セミナー，チュートリアル，講習，講義等  

開催地：Center for research and education on drug discovery, Hokkaido University, Sapporo   国名：日本国  

A complete description of the molecular and energetic factors present in biomolecular recognition phenomena mediated by proteins will facilitate the rational design of more advanced therapeutic approaches involving antibodies (and their derivatives). Numerous studies have described, in great detail, the structural, kinetic, and thermodynamic properties in the interaction between antibodies and their antigens. However, far less is known about the initial contacts and subsequent transition state leading to the formation of stable antibody-antigen complexes. By using the Eyring formalism, we have addressed this problem from a thermodynamic perspective using two different antibody-antigen systems. First, we describe recognition of MCP-1 (monocyte chemotactic protein-1), an important therapeutic target in inflammatory diseases, by the mature antibody 11K2. Mutagenesis analysis, inspired by computational calculations, demonstrated that basic thermodynamic parameters in the transition state are correlated with the improvement of affinity. Secondly, we examined a special group of antibodies vigorously studied as anti-infectious agents in the fight against AIDS. The broadly neutralizing antibody 4E10 recognizes the MPER (membrane-proximal external region) epitope in a very different environment, the membrane interface, still the thermodynamic signature at the transition state is similar to that of 11K2. The generality of our observations was evaluated independently in a different model system. We conclude that improving intermolecular interactions in the transition state may translate in higher affinity and durability of the binding complexes.

開催年月日： 2016年11月

記述言語：英語   会議種別：公開講演，セミナー，チュートリアル，講習，講義等  

開催地：Puerto Natales   国名：チリ共和国  

開催年月日： 2016年11月

記述言語：英語   会議種別：公開講演，セミナー，チュートリアル，講習，講義等  

開催地：Faculty of Engineering, Pontifical Catholic University of Chile, Santiago de Chile   国名：チリ共和国  

開催年月日： 2016年9月 - 2017年9月

記述言語：英語   会議種別：公開講演，セミナー，チュートリアル，講習，講義等  

開催地：Graduate School of Pharmaceuticl Sciences, Kyushu University, Fukuoka   国名：日本国  

The vast majority of biochemical processes in the cell involve, at some level, an intimate contact between the participating molecules. Proteins are key contributors in many molecular recognition events by directly interacting with other protein partners or with some other biomolecules such as DNA, lipids or carbohydrates. These recognition phenomena are based on carefully orchestrated non-covalent interactions comprising atoms from the protein, the partner molecule, and also from the solvent. Our experimental design employs a mixture of high-resolution techniques including X-ray crystallography, titration and scanning calorimetry, and surface plasmon resonance. We obtain a detailed structural and energetic understanding of recognition phenomena in vitro, whose implications are evaluated by complementary biological and biochemical assays. In this presentation, I will briefly summarize our contributions toward the clarification of several interaction phenomena involving proteins of therapeutic interest, such as the mechanism of action of certain anti-HIV-1 broadly-neutralizing antibodies, the recognition of antibodies by cellular receptors, the lipid-mediated metamorphosis of pore forming proteins, and the discovery and optimization of the first generation of inhibitors for the Parkinson’s disease associated protein DJ-1 and the antimicrobial target CapF. On the basis of this presentation, we are actively seeking novel directions in our research through collaboration with other fellow scientists.

開催年月日： 2016年6月

記述言語：英語   会議種別：公開講演，セミナー，チュートリアル，講習，講義等  

開催地：The Royal Society, London   国名：グレートブリテン・北アイルランド連合王国(英国)  

Pore-forming toxins (PFT) constitute a fascinating group of proteins belonging to the molecular offensive and defensive machinery of virtually all kingdoms of life. This class of water-soluble proteins shares the remarkable ability to metamorphose in the presence of cell membranes, generating lytic pores and causing cell-damage. Actinoporins are a family of potent hemolytic toxins from sea anemone forming alpha-helical pores on cellular and model membranes. In general, two requirements are sufficient to trigger pore-formation by actinoporins: (i) the presence of the lipid sphingomyelin, and (ii) the segregation of the membrane on domains or lipid-rafts. Until recently, the molecular basis of pore-formation by actinoporins, and specially the specific requirement for sphingomyelin were unclear. However, a number of recent studies have shed light into critical steps of their mechanism of action, such as binding of the toxins to the membrane, self-assembly via protein-protein interactions, and assembly of the transmembrane pore. Collectively, the data suggests that sphingomyelin facilitates pore-formation at the binding and assembly stages, and reveal the first example of a hybrid lipid/protein pore by a PFT. The structural and thermodynamic basis of this novel architecture will be explained in detail during this presentation. Surprisingly, the entire process can be made reversible under mild experimental conditions by the careful selection of detergents, challenging current perceptions in the field of membrane-protein interactions.

その他リンク： https://royalsociety.org/science-events-and-lectures/2016/06/membrane-pores/

開催年月日： 2016年3月

記述言語：英語   会議種別：公開講演，セミナー，チュートリアル，講習，講義等  

開催地：School of Engineering, The University of Tokyo, Tokyo   国名：日本国  

Pore-forming toxins (PFT) are water-soluble proteins that possess the remarkable ability to self-assemble on the membrane of target cells, where they form pores causing cell damage. We have elucidated the mechanism of action of the haemolytic PFT fragaceatoxin C (FraC), by determining crystal structures at different stages of the lytic mechanism, namely the water-soluble state, the monomeric lipid-bound form, an assembly intermediate and the fully assembled transmembrane pore. The structure of the transmembrane pore exhibits a unique architecture composed of both protein and lipids, with some of the lipids lining the pore wall, acting as assembly cofactors. The pore also exhibits lateral fenestrations that expose the hydrophobic core of the membrane to the aqueous environment. The incorporation of lipids from the target membrane within the structure of the pore provides a membrane-specific trigger for the activation of a haemolytic toxin. In addition, we describe the use of detergents with different physicochemical properties to achieve the spontaneous conversion of transmembrane pores of FraC back into the initial water-soluble state, challenging the Anfinsen dogma. Thermodynamic and kinetic stability data suggest that specific detergents cause an asymmetric change in the energy landscape of the protein, allowing the bidirectional transformation of a membrane protein. Our studies also have unveiled promising candidates for the development of protein-based biosensors highly sensitive to the concentration of cholesterol in biological membranes.

開催年月日： 2015年12月

記述言語：英語   会議種別：公開講演，セミナー，チュートリアル，講習，講義等  

開催地：Hawaii Convention Center, Honolulu, HI   国名：アメリカ合衆国  

The capsular polysaccharide (CP) forms a mucous layer on the outer surface of pathogenic Staphylococcus aureus critical for bacterial resistance. Because of the key role of CP in pathogenesis, the discovery of inhibitors targeting enzymes of the biosynthetic pathway of CP may lead to new classes of antibacterial compounds. But before getting there, it is necessary to gain a fundamental understanding of the structure, function, and mechanism of these enzymes. In this presentation I will discuss the structure/function/energetics of the coupled enzymes CapE and CapF that, together with CapG, are responsible for the synthesis of the CP precursor UDP-L-FucNAc. CapE belongs to a new subfamily of SDR enzymes present only in pathogenic strains of Gram-positive bacteria. CapE is characterized by a unique catalytic machinery and a highly dynamic hexameric organization [1,2]. Remarkably CapE undergoes large conformational changes in each catalytic cycle involving ~30% of its residues and also the cofactor NADP+. These conformational changes seem critical to generate the kinetic product instead of the more stable thermodynamic product. The enzyme CapF belongs to a new class of bifunctional enzyme composed of a metallo-cupin domain fused to a SDR domain [3]. The new crystal structure of CapF and the mechanistic data revealed the order and location of the chemical reactions occurring in each domain. We also identified unambiguously the metal ion in the cupin domain as Zn2+. The metal ion has crucial roles not only in catalysis, but also in the overall stability of the protein. Finally, I will describe our ongoing efforts to generate novel antibacterial inhibitors using fragment-based methodologies [4]. [1]Miyafusa et al. (2013) Biosci. Rep. 33:463-474, DOI: 10.1042/BSR20130017 [2]Miyafusa et al. (2013) FEBS Lett. 587:3824-3830, DOI: 10.1016/j.febslet.2013.10.009 [3]Miyafusa et al. (2011) Biochem. J. 443:671-670, DOI: 10.1042/BJ20112049 [4]Nakano et al., unpublished observations.

その他リンク： https://pacifichem.org/general-information/pacifichem-2015-interactive-pdf-program-book/

開催年月日： 2015年12月

記述言語：英語   会議種別：公開講演，セミナー，チュートリアル，講習，講義等  

国名：アメリカ合衆国  

Fragment-based drug discovery (FBDD) has enjoyed increasing popularity in academia during the screening stage of numerous drug discovery campaigns. However, the small size of the chemical compounds used in FBDD requires not only more sensitive instruments, but also novel approaches to extract quality data using fewer resources. In this presentation I will introduce SITE (single-injection thermal extinction), a thermodynamic methodology based on a modification of the well-known technique of isothermal-titration calorimetry (ITC). SITE is a fast calorimetric competitive assay suitable for automation that captures the essence of ITC but using significantly less time and fewer resources. In this presentation I will describe the principles and advantages of this methodology with three successful examples, the enzymes ketosteroid isomerase, MAP kinase ERK2, and carbonic anhydrase II. The data obtained with SITE is also evaluated versus a panel of other screening methodologies based on biophysical principles. We propose SITE as a useful tool in the identification of high-quality hits with high enthalpic efficiency in the early stages of drug discovery, and in particular that of FBDD. Reference Kobe et al. (2013) J. Med. Chem. 56:2155-2159, DOI: 10.1021/jm301603n

その他リンク： https://pacifichem.org/general-information/pacifichem-2015-interactive-pdf-program-book/

開催年月日： 2015年10月

記述言語：英語   会議種別：公開講演，セミナー，チュートリアル，講習，講義等  

開催地：Miel Park Hotel, Sendai, Miyagi   国名：日本国  

その他リンク： http://www.biophys.jp/news/lib/newsshow.php/3146

開催年月日： 2014年5月

記述言語：英語   会議種別：公開講演，セミナー，チュートリアル，講習，講義等  

開催地：ICC Jeju, Jeju Island   国名：大韓民国  

There is an increasing need to develop more precise methods in the process of drug discovery. In this regard, fragment-based drug discovery (FBDD) has enjoyed increasing popularity as screening method in the early stages of drug discovery. The small size of the fragments used in FBDD requires not only more sensitive instruments, but also novel approaches to extract quality data using fewer resources. In this presentation I will introduce SITE (single-injection thermal extinction), a thermodynamic methodology that selects high-quality hits early in FBDD [1]. SITE is a fast calorimetric competitive assay suitable for automation that captures the essence of isothermal titration calorimetry but using significantly less time and materials. In this presentation I will describe the principles and advantages of this methodology with practical examples.

その他リンク： http://www.pssj.jp/APPA/activity.html

開催年月日： 2013年2月

記述言語：英語   会議種別：公開講演，セミナー，チュートリアル，講習，講義等  

開催地：The University of Tokyo, Tokyo   国名：日本国  

開催年月日： 2012年9月

記述言語：英語   会議種別：シンポジウム・ワークショップ パネル（公募）  

開催地：Yokohama City University, Yokohama   国名：日本国  

開催年月日： 2012年4月

記述言語：日本語   会議種別：公開講演，セミナー，チュートリアル，講習，講義等  

国名：日本国  

開催年月日： 2011年12月

記述言語：英語   会議種別：公開講演，セミナー，チュートリアル，講習，講義等  

開催地：Institute of Medical Science, The University of Tokyo, Tokyo   国名：日本国  

Proper functioning of biological systems demands exquisite recognition of binding partners at the molecular level. How to achieve such high specificity in the complex environment of the cell? Intensive research in this field has revealed the physicochemical nature of many of the forces involved in recognition phenomena, such as in antigen-antibody, ligand-receptor, or enzyme-substrate complexes. This detailed knowledge has facilitated the design of novel and better drugs to combat human disease. However, there remains a large pool of recalcitrant “transient” associations that are still poorly understood. Our next grand challenge consists in the detailed mapping, cataloging, and biophysical characterization of weak and ultraweak biomolecular interactions. These investigations are expanding at a rapid pace thanks to technological and conceptual innovations in multiple fronts. In this presentation, I will discuss two biological systems displaying distinctive weak and/or ultraweak binding properties: (1) lipid-protein, and (2) protein-sugar interactions. In the first example I will review mechanisms by which lipid composition may modulate the optimal activity of membrane proteins and lytic peptides. In addition, I will discuss how pore-forming proteins may exploit small physical imperfections on the surface of the membrane to induce cell lysis. In the second example, I will introduce a sugar-processing enzyme (GalM) as a model system to study ultraweak protein-sugar interactions. In this study we have deepened our understanding about the nature of C-Hπ interactions in modulating sugar recognition and binding affinity. Overall, our data supports an increasingly important role of aromatic interactions and water-mediated contacts during transient biomolecular associations. Future challenges will be discussed if time permits.

開催年月日： 2011年9月

記述言語：英語   会議種別：公開講演，セミナー，チュートリアル，講習，講義等  

開催地：Bangkok   国名：日本国  

The emergence and proliferation of bacteria carrying antibiotic resistance is a serious problem that threatens public health. This is particularly grave in certain multi-resistant strains of bacterium Staphylococcus aureus (so-called superbug). In order to sustain effective infection, this bacterium steals the iron atom contained in the heme group of hemoglobin from the host organism. The heme acquisition system of S. aureus is composed by more than ten proteins collectively known as Iron-regulated surface determinant (Isd). Isd proteins are strategically located all the way from the cell surface to the interior of the cell. Heme molecules are transferred among Isd proteins in an orderly and sequential fashion. Blocking this critical iron supply-route in S. aureus is an attractive therapeutic possibility that may alleviate disease and suffering. However, we must investigate the molecular basis of heme transfer within Isd before this therapeutic approach can be brought to fruition. We are particularly interested in understanding (i) the extent of binding of heme to Isd proteins, (ii) the nature of protein-protein interactions among Isd members, and (iii) the thermodynamic and structural factors underlying these complex interactions. We brought together isothermal titration calorimetry, UV-visible spectrometry, circular dichroism, site-specific mutagenesis, and high-resolution X-ray crystallography to understand the energetic and dynamic properties controlling the first stages of heme acquisition. We found out that the initial stages in the internalization route require binding of heme ligand to receptors with high affinity. We also observed that transfer of heme towards the interior of the cell is fast and largely irreversible, thus avoiding back-transfer reaction and limiting the loss of precious heme molecules back to solution. Our results also suggested that heme transfer is facilitated by specific protein-protein interactions between sequential Isd receptors. Moreover, by using non-iron metalloporphyrins (i.e. the equivalent of heme but containing a metal other than iron) we demonstrated that electrostatic forces mediate recognition of ligand. X-ray crystal structures of receptor IsdH with heme and other non-iron porphyrins clarified some of the energetic attributes determined in our thermodynamic analyses. We hope our study will help to develop new therapeutic agents against S. aureus.

開催年月日： 2010年12月

記述言語：英語   会議種別：公開講演，セミナー，チュートリアル，講習，講義等  

開催地：Hawaii Convention Center, Honolulu, HI   国名：アメリカ合衆国  

Protein-carbohydrate interactions regulate numerous molecular recognition events, such as bacterial and viral infections, cell adhesion in inflammation and metastasis, and many other intracellular and signal transduction pathways. Although numerous crystals structures of carbohydrate binding proteins are available in the Protein Data Bank, there remain conflicting views about the exact nature of the forces involved in carbohydrate binding and recognition. In this study we aimed at understanding the nature of the forces involved in the binding of galactose glucose to the model enzyme Galactose Mutarotase (GalM) from Escherichia coli. First, the crystal structure of GalM with Galactose bound was solved by the Multiwavelength Anomalous Diffraction (MAD) method, and the phases extended to a final resolution of 1.4 Å. E. coli GalM forms a β-sandwich that closely resembles that of the homolog enzyme from Lactobacillus lactis [1]. The active site displays numerous polar residues that orient the substrate towards catalytic residues Glu309, His104 and His175. Substitution of these residues did not significantly alter the architecture of the binding site, while dramatically diminished the enzymatic activity. Aromatic residue Tyr284 is located at the bottom of the binding site towards the alpha-face of the galactose moiety. High-resolution X-ray crystallography, Isothermal Titration Calorimetry, and enzymatic assays performed with wild type and Tyr284-substituted proteins revealed that the presence of an aromatic residue at this position is essential for binding of the sugar moiety, although the substitutions do not completely abolish the activity of the enzyme. More importantly, this effect is exclusively of aromatic nature, since hydrophobic residues at that position do not promote binding of the sugar.

開催年月日： 2007年12月

記述言語：日本語   会議種別：公開講演，セミナー，チュートリアル，講習，講義等  

開催地：Department of Structural Biology, Graduate School of Pharmaceutical Sciences, Kyoto University, Kyoto   国名：日本国  

開催年月日： 2002年8月

記述言語：日本語   会議種別：公開講演，セミナー，チュートリアル，講習，講義等  

開催地：Boston, MA   国名：アメリカ合衆国  

開催年月日： 2002年1月

記述言語：英語   会議種別：公開講演，セミナー，チュートリアル，講習，講義等  

開催地：Department of Biochemistry and Molecular Biology, UPV/EHU, Bilbao   国名：スペイン  

開催年月日： 2002年1月

記述言語：英語   会議種別：公開講演，セミナー，チュートリアル，講習，講義等  

開催地：Department of Materials Science, University of Zaragoza, Zaragoza   国名：スペイン  

開催年月日： 2001年12月

記述言語：英語   会議種別：シンポジウム・ワークショップ パネル（公募）  

開催地：Madrid   国名：スペイン  

開催年月日： 2001年9月

記述言語：英語   会議種別：公開講演，セミナー，チュートリアル，講習，講義等  

開催地：Portoroz   国名：スロベニア共和国  

その他リンク： http://bio.ijs.si/SBD/partic99.htm

記述言語：日本語  

記述言語：英語   掲載種別：機関テクニカルレポート，技術報告書，プレプリント等  

その他リンク： http://pfwww.kek.jp/acr/2018pdf/u_reports/pf18b0243.pdf

記述言語：英語   掲載種別：機関テクニカルレポート，技術報告書，プレプリント等  

その他リンク： http://pfwww.kek.jp/acr/2018pdf/u_reports/pf18b0242.pdf

記述言語：英語   掲載種別：機関テクニカルレポート，技術報告書，プレプリント等  

その他リンク： http://pfwww.kek.jp/acr/2017pdf/u_reports/pf17b0100.pdf

記述言語：日本語   掲載種別：記事・総説・解説・論説等（学術雑誌）  

In small molecule drug discovery, researchers must find specific binders that interact with a target protein and inhibit its function in connection with human diseases. It is of critical importance to know the binding mode of com- pounds interacting with a target protein to assure hit validation and optimization. Biophysical analysis is a powerful quantitative approach to evaluate the binding modes of such candidates. Since the level of sensitivity of biophysical analysis is suitable to quantitatively detect the binding of fragment compounds, and because of the remarkable success of compound libraries of small molecules, the development and adaptation of biophysical analysis for these applications is in great demand. Herein, we describe the technical developments of biophysical methods, especially thermodynamic and kinetic analysis, for the purpose of screenings which employ small molecules. In addition, we discuss the interaction mechanisms of small molecules to find hit compounds based on these biophysical analyses.

DOI： 10.1248/yakushi.17-00211-2

その他リンク： https://www.jstage.jst.go.jp/article/yakushi/138/8/138_17-00211-2/_article/-char/ja

記述言語：英語   掲載種別：機関テクニカルレポート，技術報告書，プレプリント等  

その他リンク： http://pfwww.kek.jp/acr/2017pdf/u_reports/pf17b0103.pdf

記述言語：英語   掲載種別：機関テクニカルレポート，技術報告書，プレプリント等  

その他リンク： http://pfwww.kek.jp/acr/2017pdf/u_reports/pf17b0102.pdf

記述言語：日本語   掲載種別：記事・総説・解説・論説等（学術雑誌）  

黄色ブドウ球菌が持つ鉄取り込み機構 Isd 系は，きわめて微弱な相互作用を利用する絶妙な タンパク質−タンパク質間認識，複数の担体によるヘムの細胞壁中でのリレー輸送，新規 なヘム結合環境，そして新規な化学作用を有する異化酵素など，数多くの興味深い特性を 包含している一連のタンパク質群によって担われる.本稿では Isd システムの構造・物性解 析に関する報告から明らかになった，Isd および他の同族体のヘム輸送系に関する分子機構 をまとめ，本系を治療標的とする可能性等，今後の展開を議論したい.

DOI： 10.14952/SEIKAGAKU.2018.900279

その他リンク： https://seikagaku.jbsoc.or.jp/10.14952/SEIKAGAKU.2018.900279/index.html

記述言語：英語   掲載種別：記事・総説・解説・論説等（学術雑誌）  

ntibodies of the IgG class carry a pair of oligosaccharides (N-glycans) in the Fc region. The importance of the N-glycan is clearly demonstrated by the profound effect it has in the physicochemical and biological properties of antibodies. The term ‘glycoengineering’ has been coined to describe contemporary strategies to improve the performance of therapeutic monoclonal antibodies on the basis of modi cations in the structure and composition of the N-glycan. These methodologies have resulted in the approval and commercialization of a new generation of antibodies with improved therapeutic efficacy. So far, these advances have been driven by herculean efforts in a process of trial-and-error. The collective work of researchers in this eld is progressively revealing the molecular basis of N-glycans for the function of antibodies. This knowledge will ultimately be conducive to the application of rational approaches for the successful manipulation of antibodies using glycoengineering strategies. Herein,
we review advances in our understanding of the role of the N-glycan in the structural and dynamic integrity, and biological activity, of antibodies. Since the N-glycan has a multifaceted effect in antibodies, in this review we have emphasized the importance of integrating various techniques that address
this problem from multiple points of view. In particular, the combination of X-ray crystallography, with nuclear magnetic resonance, molecular dynamics simulations and biophysical approaches based on thermodynamic principles, has emerged as a powerful combination that is deepened our understanding of this unique system with critical implications for human well-being.

その他リンク： https://doi.org/10.1093/intimm/dxx038

記述言語：英語   掲載種別：機関テクニカルレポート，技術報告書，プレプリント等  

Fragaceatoxin C (FraC) is a pore forming toxin (PFT) that interacts with biological membranes using a lipid binding motif. Since FraC also interact with the carbohydrates, we investigated the basis of glycan recognition. Surprisingly, we found that FraC engages glycans using the same, high-affinity, lipid-binding module. In conclusion, FraC has developed dual recognition capabilities with a single binding motif.

その他リンク： http://pfwww.kek.jp/acr/2016pdf/u_reports.html

記述言語：英語   掲載種別：機関テクニカルレポート，技術報告書，プレプリント等  

The mechanism by which the MPER epitope of gp41 of HIV-1 is recognized by the broadly neutralizing antibody 10E8 at membrane interfaces is not known. We determined the crystal structure of 10E8 Fab complex in complex with the transmembrane antigen in detergents. Our research clarifies the molecular mechanisms underlying broad neutralization by 10E8, and guiding future vaccination strategies targeting the MPER antigen of HIV-1.

その他リンク： http://pfwww.kek.jp/acr/2016pdf/u_reports.html

記述言語：英語   掲載種別：記事・総説・解説・論説等（学術雑誌）  

Antibodies are a family of glycoproteins that bind specifically to target molecules (antigens). The antibody-binding sites are formed by six segments of variable structure (CDRs) supported by a scaffold of essentially invariant architecture (frame-work regions). Shape complementarity between the contact surfaces (in the case of protein antigens) or close interactions with small antigens (hapten, peptide or others), together with complementation of water molecules, are important to achieve high affinity and specificity. The binding of an antigen to an antibody takes place by the formation of multiple noncovalent bonds between the antigen and the amino acids of the binding site. The increase in van der Waals contacts and/or buried surfaces upon complexation generally correlates well with the binding strength. Hydrogen bonds are in most cases critical to achieve high specificity and affinity for the antigen target. Importantly, antibodies have at least two antigen binding sites, boosting the effective affinity of the antibody for its target by a mechanism termed avidity.

その他リンク： http://onlinelibrary.wiley.com/doi/10.1002/9780470015902.a0001117.pub3/abstract

記述言語：英語  

Receptors of the Fc region of immunoglobulin-G (IgG) are key mediators of the immune response. In particular, human Fcgamma receptor I (hFcgammaRI) is the immune receptor with the highest affinity for IgG. To understand the molecular basis of interaction with antibodies, we determined the crystal structure of the complex between hFcgammaRI and human IgG-Fc at high resolution (1.80 Å). The structure reveals a deep and hydrophobic pocket explaining the strong affinity for IgG an- tibodies. We propose a general model for binding of IgG to Fc receptors on the cell surface. Our findings have implications for the development of novel therapeutic approaches involving hFcgammaRI.

その他リンク： http://www2.kek.jp/imss/pf/science/publ/pfhl/2015/

記述言語：英語   掲載種別：機関テクニカルレポート，技術報告書，プレプリント等  

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その他リンク： http://pfwww.kek.jp/acr/2015pdf/part_b.html

記述言語：英語   掲載種別：機関テクニカルレポート，技術報告書，プレプリント等  

Not available

その他リンク： http://pfwww.kek.jp/acr/2015pdf/part_b.html

記述言語：英語   掲載種別：記事・総説・解説・論説等（学術雑誌）  

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その他リンク： http://jvi.asm.org/content/90/6/3276.full

記述言語：英語   掲載種別：記事・総説・解説・論説等（学術雑誌）  

The number of studies and the quality of the structural data of Fcgamma receptors (FcgammaRs) has rapidly increased in the last few years. Upon critical examination of the literature, we have extracted general conclusions that could explain differences in affinity and selectivity of FcgammaRs for immunoglobulin G (IgG) based on structural considerations. FcgammaRs employ a little conserved asymmetric surface of domain D2 composed of two distinct subsites to recognize the well-conserved lower hinge region of IgG1-Fc. The extent of the contact interface with the antibody in subsite 1 of the receptor (but not in subsite 2), the geometrical complementarity between antibody and receptor, and the number of polar interactions contribute decisively toward strengthening the binding affinity of the antibody for the receptor. In addition, the uncertain role of the N-linked glycan of IgG for the binding and effector responses elicited by FccRs is discussed. The available data suggest that not only the non-covalent interactions between IgG and FccRs but also their dynamic features are essential for the immune response elicited through these receptors. We believe that the integration of structural, thermodynamic, and kinetic data will be critical for the design and validation of the next generation of therapeutic antibodies with enhanced effector capabilities.

その他リンク： http://onlinelibrary.wiley.com/doi/10.1111/imr.12365/abstract

記述言語：日本語   掲載種別：機関テクニカルレポート，技術報告書，プレプリント等  

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その他リンク： http://pfwww.kek.jp/acr2013pdf/part_b.html

記述言語：日本語   掲載種別：機関テクニカルレポート，技術報告書，プレプリント等  

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その他リンク： http://pfwww.kek.jp/acr2013pdf/part_b.html

記述言語：日本語   掲載種別：機関テクニカルレポート，技術報告書，プレプリント等  

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その他リンク： http://pfwww.kek.jp/acr2013pdf/part_b.html

記述言語：日本語  

FraC はイソギンチャクが分泌する Pore-Forming Toxin である。Pore-Forming Toxin は可溶性蛋白質として発現した後, 生体膜に付着して膜貫通蛋白質に変化するという興味深い挙動を示す。本研究では,FraC の単量体と反応中間体構造の構 造比較,及び生体膜の組成が FraC の小孔形成に及ぼす影響の解析を通じて,FraC の詳細な反応機構を提案した。FraC の 有する膜貫通ドメインは生体膜の小さなゆるみを感知するセンサーであることが示され,FraC の多量体化に伴う「疎水ポ ケット挿入残基の交換反応」がセンサー作動のスイッチとなることが示唆された。

その他リンク： http://pfwww.kek.jp/publications/pfnews/31_2/31_2.html

記述言語：英語   掲載種別：機関テクニカルレポート，技術報告書，プレプリント等  

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その他リンク： http://pfwww.kek.jp/acr2010pdf/part_b.html

記述言語：英語   掲載種別：機関テクニカルレポート，技術報告書，プレプリント等  

Not available

その他リンク： http://pfwww.kek.jp/acr2010pdf/part_b.html

記述言語：英語   掲載種別：機関テクニカルレポート，技術報告書，プレプリント等  

Not available

その他リンク： http://pfwww.kek.jp/acr2010pdf/part_b.html

記述言語：英語   掲載種別：機関テクニカルレポート，技術報告書，プレプリント等  

Not available

その他リンク： http://pfwww.kek.jp/acr2010pdf/part_b.html

種別：査読等 

外国語雑誌　査読論文数：9

日本語雑誌　査読論文数：0

国際会議録　査読論文数：0

国内会議録　査読論文数：0

種別：査読等 

外国語雑誌　査読論文数：9

日本語雑誌　査読論文数：0

国際会議録　査読論文数：0

国内会議録　査読論文数：0

種別：大会・シンポジウム等 

参加者数：40

種別：大会・シンポジウム等 

参加者数：45

種別：大会・シンポジウム等 

参加者数：50

種別：学会・研究会等 

種別：大会・シンポジウム等 

参加者数：40

種別：大会・シンポジウム等 

参加者数：250