Kyushu University Academic Staff Educational and Research Activities Database
List of Papers
Caaveiro Jose Last modified date:2022.01.05

Professor / Faculty of Pharmaceutical Sciences


Papers
1. Rujas, E., Leaman, D.P., Insausti, S., Carravilla, P., García-Porras, M., Largo, E., Morillo, I., Sanchez-Eugenia, R., Zhang, L., Cui, H., Iloro, I., Elortza, F., Julien, J.-P., Eggeling, C., Zwick, M.B., Caaveiro, J.M.M.*, and Nieva, J.L. , Focal accumulation of aromaticity at the CDRH3 loop mitigates 4E10 polyreactivity without altering its HIV neutralization profile, iScience, 10.1016/j.isci.2021.102987, 24, 102987, 2021.09, [URL], 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.
.
2. Akiba, H., Tamura, H., Caaveiro, J.M.M.*, and Tsumoto, K. , Epitope-dependent thermodynamic signature of single-domain antibodies against hen egg lysozyme, J. Biochem., 10.1093/jb/mvab082, 2021.09, [URL], 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..
3. Yui, A., Caaveiro, J.M.M.*, Kuroda, D., Nakakido, M., Nagatoishi, S., Goda, S., Maruno, T., Uchiyama, S., and Tsumoto, K., Mechanism of dimerization and structural features of human LI-cadherin, J. Biol. Chem., 10.1016/j.jbc.2021.101054, 297, 101054, 2021.08, [URL], 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..
4. Kosuke Oyama, Takatoshi Ohkuri, Jinta Ochi, Jose M.M. Caaveiro, Tadashi Ueda, Abolition of aggregation of CH2 domain of human IgG1 when combining glycosylation and protein stabilization, Biochim. Biophyys. Res. Commun., https://doi.org/10.1016/j.bbrc.2021.04.070, 2021.04, [URL], 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..
5. Kosuke Oyama, Takatoshi Ohkuri, Mao Inoue, Jose M.M. Caaveiro, Tadashi Ueda1, High-level expression of human CH2 domain from the Fc region in Pichia pastoris and preparation of anti-CH2 antibodies, J. Biochem., doi:10.1093/jb/mvab039, 2021.03, [URL], 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..
6. Tomohiro Yamashita, Sawako Kamikaseda, Aya Tanaka, Hidetoshi Tozaki-Saitoh, Jose M. M. Caaveiro, Kazuhide Inoue and Makoto Tsuda, New Inhibitory Effects of Cilnidipine on Microglial P2X7 Receptors and IL-1 Release: An Involvement in its Alleviating Effect on Neuropathic Pain, Cells, https://doi.org/10.3390/cells10020434, 2021.02, [URL], 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.
.
7. Misaki Ishii, Makoto Nakakido, Jose M.M. Caaveiro, Daisuke Kuroda, C.J. Okumura, Toshiaki Maruyama, Kevin Entzminger, Kouhei Tsumoto, Structural basis for antigen recognition by methylated lysine–specific antibodies, J. Biol. Chem., https://doi.org/10.1074/jbc.RA120.015996, 2020.12, [URL], 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..
8. Edurne Rujas, Sara Insausti, Daniel P. Leaman, Pablo Carravilla, Saul Gonzalez-Resines, Valerie Monceaux,Ruben Sanchez-Eugenia, Miguel Garcıa-Porras, Ibon Iloro, Lei Zhang, Felix Elortza, Jean-Philippe Julien, Asier Saez-Cirion, Michael B. Zwick, Christian Eggeling, Akio Ojida, Carmen Domene, Jose M.M. Caaveiro, and Jose L. Nieva, Affinity for the Interface Underpins Potency of Antibodies Operating In Membrane Environments, Cell Reports, https://doi.org/10.1016/j.celrep.2020.108037, 32, 108037, 2020.08, [URL], 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..
9. H. Akiba, H., H. Tamura, J.M.M. Caaveiro, and K. Tsumoto, Computer-guided library generation applied to the optimization of single-domain antibodies, Prot. Eng. Des. Select., 10.1093/protein/gzaa006, 32, 9, 423-431, 2020.03, [URL], 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..
10. Akiba, H., Tamura, H., Kiyoshi, M., Yanaka, S., Sugase, K., Caaveiro, J.M.M.,* Tsumoto, K, Structural and thermodynamic basis for the recognition of the substrate-binding cleft on hen egg lysozyme by a single-domain antibody, Sci. Rep., 10.1038/s41598-019-50722-y 11, 9, 15481, 2019.10, [URL], 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..
11. Morante, K., Bellomio, A., Viguera, A.R., González-Mañas, J.M., Tsumoto, K., Caaveiro, J.M.M.*, The Isolation of New Pore-Forming Toxins from the Sea Anemone Actinia fragacea Provides Insights into the Mechanisms of Actinoporin Evolution, Toxins, 10.3390/toxins11070401, 11, 2019.07, [URL], 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..
12. Yamamoto, S., Yamashita, T., Ito, M., Caaveiro, J.M.M., Egashira, N., and Tsuda, M., New pharmacological effect of fulvestrant to prevent oxaliplatin-induced peripheral neuropathy in rats., Int. J. Cancer, 10.1002/ijc.32043, 145, 2107-2113, 2019.07, [URL], 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..
13. Shindo, N., Fuchida, H., Sato, M., Watari, K., Shibata, T., Kuwata, K., Miura, C., Okamoto, K., Hatsuyama, Y., Tokunaga, K., Sakamoto, S., Morimoto, S., Abe, Y., Shiroishi, M., Caaveiro, J.M.M., Ueda, T., Tamura, T., Matsunaga, N., Nakao, T., Odo, S., Yamaguchi, Y., Hamachi, I., Ono, M., and Ojida A., Selective and reversible covalent modification of non-catalytic cysteines with weakly reactive α-chlorofluoroacetamides., Nat. Chem. Biol., 10.1038/s41589-018-0204-3, 2019.01, [URL], 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..
14. Tashiro, S., Caaveiro, J.M.M.,* Tanabe, A., Nagatoishi, S., Tamura, Y., Matsuda, N., Liu, D., Hoang, Q.Q., and Tsumoto, K, Discovery and optimization of potent inhibitors of the Parkinson’s disease associated protein DJ-1, ACS Chem. Biol, 10.1021/acschembio.8b00701, 2018.07, [URL], 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..
15. Miyanabe, K., Akiba, H., Kuroda, D., Nakakido, M., Kusano-Arai, O., Iwanari, H., Hamakubo, T., Caaveiro, J.M.M.,* and Tsumoto, K., Intramolecular H-bonds govern the recognition of a flexible peptide by an antibody., J. Biochem., 10.1093/jb/mvy032, 164, 65-76, 2018.07, [URL], 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.
.
16. Miyanabe, K., Yamashita, T., Akiba, H., Takamatsu, Y., Nakakido, M., Hamakubo, T., Caaveiro, J.M.M.,* and Tsumoto, K., Tyrosine Sulfation Restricts the Conformational Ensemble of a Flexible Peptide, Strengthening the Binding Affinity for an Antibody, Biochemistry, 10.1021/acs.biochem.8b00592, 57, 4177-4185, 2018.06, [URL], 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..
17. Tashima, T., Nagatoishi, S., Caaveiro, J.M.M., Nakakido, M., Sagara, H., Mimuro, H., Ohnuma, S-I., and Tsumoto, K., Weak electrostatic interactions between collagen and monomeric SLRP osteomodulin govern the shape of type I collagen fibrils, Commun. Biol., 10.1038/s42003-018-0038-2, 1, 33, 2018.04, [URL], 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..
18. Kiyoshi, M., Caaveiro, J.M.M., Tada, M., Tamura, H., Tanaka, T., Terao, Y., Morante, K., Harazono, A., Hashii, N., Shibata, H., Kuroda, D., Nagatoishi, S., Oe, S., Ide, T., Tsumoto, K., and Ishii-Watabe, A., Assessing the heterogeneity of the Fc-glycan of a therapeutic antibody using an engineered FcγReceptorIIIa-immobilized column., Sci. Rep., 10.1038/s41598-018-22199-8, 8, 3955, 2018.03, [URL], 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..
19. Tanaka, K., Caaveiro, J.M.M.,* Morante, K., Tsumoto, K., Hemolytic actinoporins interact with carbohydrates using their lipid-binding module, Philos. Trans. R. Soc. Lond. B Biol. Sci., 10.1098/rstb.2016.0216, 372, 20160216, 2017.06, [URL], 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..
20. Rujas, E., Insausti, S., Garcia-Porras, M., Sanchez-Eugenia, R., Tsumoto, K., Nieva, J.L., Caaveiro, J.M.M.*, Functional contacts between MPER and the anti-HIV-1 broadly neutralizing antibody 4E10 extend into the core of the membrane., J. Mol. Biol., 10.1016/j.jmb.2017.03.008, 429, 1213-1226, 2017.04, [URL], 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..
21. Rujas, E., Caaveiro, J.M.M., Insausti, S., Garcia-Porras, M., Tsumoto, K., Nieva, J.L., Peripheral Membrane Interactions Boost the Engagement by an Anti HIV-1 Broadly Neutralizing Antibody, J. Biol. Chem., 10.1074/jbc.M117.775429, 292, 5571-5583, 2017.03, [URL], 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..
22. Kudo, S., Caaveiro, J.M.M., Nagatoishi, S., Miyafusa, M., Matsuura, T., Sudou, Y., Tsumoto, K., Disruption of cell adhesion by an antibody targeting the cell-adhesive intermediate (X-dimer) of human P-cadherin, Sci. Rep., 10.1038/srep39518, 7, 2017.01, [URL], 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..
23. Garcia-Linares, S., Rivera-de-Torre, E., Morante, K., Tsumoto, K., Caaveiro, J.M.M., Gavilanes, J.G., Slotte, J.P., Martinez-del-Pozo, A., Differential Effect of Membrane Composition on the Pore-Forming Ability of Four Different Sea Anemone Actinoporins, Biochemistry, 10.1021/acs.biochem.6b01007, 55, 48, 6630-6641, 2016.12, [URL].
24. Rujas, E., Caaveiro, J.M.M., Partida-Hanon, A., Gulzar, N., Morante, K., Apellaniz, B., Garcia-Porras, M., Bruix, M., Tsumoto, K., Scott, J.K., Jimenez, M.A., Nieva, J.L., Structural basis for broad neutralization of HIV-1 through the molecular recognition of 10E8 helical epitope at the membrane interface, Sci. Rep., 10.1038/srep38177, 6, 2016.12, [URL], 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..
25. Morante, K., Bellomio, A., Gil-Carton, D., Redondo-Morata, L., Sot, J., Scheuring, S., Valle, M., Gonzalez-Manas, J.J., Tsumoto, K., Caaveiro, J.M.M.*, Identification of a Membrane-bound Prepore Species Clarifies the Lytic Mechanism of Actinoporins, J. Biol. Chem., 10.1074/jbc.M116.734053, 291, 37, 19210-19219, 2016.09, [URL], 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..
26. Kudo, S., Caaveiro, J.M.M.,* Tsumoto, K., Adhesive Dimerization of Human P-Cadherin Catalyzed by a Chaperone-like Mechanism, Structure, 10.1016/j.str.2016.07.002, 24, 9, 1523-1536, 2016.09, [URL], 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..
27. Sigala, P.A., Morante, K., Tsumoto, K., Caaveiro, J.M.M., Goldberg, D.E., In-Cell Enzymology To Probe His-Heme Ligation in Heme Oxygenase Catalysis, Biochemistry, 10.1021/acs.biochem.6b00562, 55, 34, 4836-4849, 2016.08, [URL], 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..
28. Rujas, E., Gulzar, N., Morante, K., Tsumoto, K., Scott, J.K., Nieva, J.L., Caaveiro, J.M.M.*, Structural and Thermodynamic Basis of Epitope Binding by Neutralizing and Nonneutralizing Forms of the Anti-HIV-1 Antibody 4E10, J. Virol., 10.1128/JVI.01793-15, 89, 23, 11975-11989, 2015.12, [URL], 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..
29. Tanaka, K., Caaveiro, J.M.M.,* Tsumoto, K., Bidirectional Transformation of a Metamorphic Protein between the Water-Soluble and Transmembrane Native States, Biochemistry, 10.1021/acs.biochem.5b01112, 54, 46, 6863-6866, 2015.11, [URL], 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..
30. Nakano, K., Chigira, T., Miyafusa, T., Nagatoishi, S., Caaveiro, J.M.M.,* Tsumoto, K., Discovery and characterization of natural tropolones as inhibitors of the antibacterial target CapF from Staphylococcus aureus, Sci. Rep., 10.1038/srep15337, 5, 2015.10, [URL], 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..
31. Morante, K., Caaveiro, J.M.M., Viguera, A.R., Tsumoto, K., Gonzalez-Manas, J.M., Functional characterization of Val60, a key residue involved in the membrane-oligomerization of fragaceatoxin C, an actinoporin from Actinia fragacea, FEBS Lett., 10.1016/j.febslet.2015.06.012, 589, 15, 1840-1846, 2015.07, [URL], 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..
32. Apellaniz, B., Rujas E., Serrano, S., Morante, K., Tsumoto, K., Caaveiro, J.M.M.,* Jimenez, MA, Nieva, J.L., The Atomic Structure of the HIV-1 gp41 Transmembrane Domain and Its Connection to the Immunogenic Membrane-proximal External Region, J. Biol. Chem., 10.1074/jbc.M115.644351, 290, 21, 12999-13015, 2015.05, [URL], 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..
33. Kiyoshi, M., Caaveiro, J.M.M.,* Kawai, T., Tashiro, S., Ide, T., Asaoka, Y., Hatayama, K., Tsumoto, K., Structural basis for binding of human IgG1 to its high-affinity human receptor Fc gamma RI, Nat. Commun., 10.1038/ncomms7866, 6, 2015.04, [URL], 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..
34. Morante, K., Caaveiro, J.M.M.,* Tanaka, K., Gonzalez-Manas, J.M., Tsumoto, K., A Pore-Forming Toxin Requires a Specific Residue for Its Activity in Membranes with Particular Physicochemical Properties, J. Biol. Chem., 10.1074/jbc.M114.615211, 290, 17, 10850-10861, 2015.04, [URL], 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..
35. Tanaka, K., Caaveiro, J.M.M.,* Morante, K., Gonzalez-Manas, J.M., Tsumoto, K., Structural basis for self-assembly of a cytolytic pore lined by protein and lipid, Nat. Commun., 10.1038/ncomms7337, 6, 2015.02, [URL], 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..
36. Tashiro, S., Caaveiro, J.M.M.,* Wu, C.-X., Hoang, Q.Q., Tsumoto, K., Thermodynamic and Structural Characterization of the Specific Binding of Zn(II) to Human Protein DJ-1, Biochemistry, 10.1021/bi500294h, 53, 14, 2218-2220, 2014.04, [URL], 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)..
37. Kudo, S., Caaveiro, J.M.M., Goda, S., Nagatoishi, S., Ishii, K., Matsuura, T., Sudou, Y., Kodama, T., Hamakubo, T., Tsumoto, K., Identification and Characterization of the X-Dimer of Human P-Cadherin: Implications for Homophilic Cell Adhesion, Biochemistry, 10.1021/bi401341g, 53, 11, 1742-1752, 2014.03, [URL], 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..
38. Kiyoshi, M., Caaveiro, J.M.M., Miura, E., Nagatoishi, S., Nakakido, M., Soga, S., Shirai, H., Kawabata, S., Tsumoto, K., Affinity Improvement of a Therapeutic Antibody by Structure-Based Computational Design: Generation of Electrostatic Interactions in the Transition State Stabilizes the Antibody-Antigen Complex, PLOS ONE, 10.1371/journal.pone.0087099, 9, 1, 2014.01, [URL], 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..
39. Moriwaki, Y., Terada, T., Caaveiro, J.M.M., Takaoka, Y., Hamachi, H., Tsumoto, K., Shimizu, K., Heme Binding Mechanism of Structurally Similar Iron-Regulated Surface Determinant Near Transporter Domains of Staphylococcus aureus Exhibiting Different Affinities for Heme, Biochemistry, 10.1021/bi4008325, 52, 49, 8866-8877, 2013.12, [URL], 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..
40. Miyafusa, T., Caaveiro, J.M.M., Tanaka, Y., Tsumoto, K., Dynamic elements govern the catalytic activity of CapE, a capsular polysaccharide-synthesizing enzyme from Staphylococcus aureus, FEBS Lett., 10.1016/j.febslet.2013.10.009, 587, 23, 3824-3830, 2013.11, [URL], 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..
41. Vu, N., Moriwaki, Y., Caaveiro, J.M.M.,* Terada, T., Tsutsumi, H., Hamachi, I., Shimizu, K., Tsumoto, K., Selective binding of antimicrobial porphyrins to the heme-receptor IsdH-NEAT3 of Staphylococcus aureus, Prot. Sci., 10.1002/pro.2276, 22, 7, 942-953, 2013.07, [URL], 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..
42. Sigala, P.A., Fafarman, A.T., Schwans, J.P., Fried, S.D., Fenn, T.D., Caaveiro, J.M.M., Pybus, B., Ringe, D., Petsko, G.A., Boxer, S.G., Herschlag, D., Quantitative dissection of hydrogen bond-mediated proton transfer in the ketosteroid isomerase active site, Proc. Natl. Acad. Sci. USA, 10.1073/pnas.1302191110, 110, 28, E2552-E2561, 2013.07, [URL], 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..
43. Kobe, A., Caaveiro, J.M.M.,* Tashiro, S., Kajihara, D., Kikkawa, M., Mitani, T, Tsumoto, K., Incorporation of Rapid Thermodynamic Data in Fragment-Based Drug Discovery, J. Med. Chem., 10.1021/jm301603n, 56, 5, 2155-2159, 2013.03, [URL], 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..
44. Miyafusa, T., Caaveiro, J.M.M.,* Tanaka, Y., Tsumoto, K., Crystal structure of the enzyme CapF of Staphylococcus aureus reveals a unique architecture composed of two functional domains, Biochem. J., 10.1042/BJ20112049, 443, 671-680, 2012.05, [URL], 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..
45. Kudo, S., Caaveiro, J.M.M., Miyafusa, T., Goda, S., Ishii, K., Matsuura, T., Sudou, Y., Kodama, T., Hamakubo, T., Tsumoto, K., Structural and thermodynamic characterization of the self-adhesive properties of human P-cadherin, Mol. Biosyst., 10.1039/c2mb25161b, 8, 8, 2050-2053, 2012.05, [URL], 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..
46. Abe, R., Caaveiro, J.M.M.,* Kozuka-Hata, H., Oyama, M., Tsumoto, K., Mapping Ultra-weak Protein-Protein Interactions between Heme Transporters of Staphylococcus aureus, J. Biol. Chem., 10.1074/jbc.M112.346700, 287, 20, 16477-16487, 2012.05, [URL], 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..
47. Miyafusa, T., Caaveiro, J.M.M., Tanaka, Y., Tanner, M.E., Tsumoto, K., Crystal structure of the capsular polysaccharide synthesizing protein CapE of Staphylococcus aureus, Biosci. Rep., 10.1042/BSR20130017, 33, 463-474, 2012.04, [URL], 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..
48. Kawai. T., Caaveiro, J.M.M.,* Abe, R., Katagiri, T., Tsumoto, K., Catalytic activity of MsbA reconstituted in nanodisc particles is modulated by remote interactions with the bilayer, FEBS Lett., 10.1016/j.febslet.2011.10.015, 585, 22, 3533-3537, 2011.11, [URL], 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..
49. Moriwaki, Y., Caaveiro, J.M.M.,* Tanaka, Y., Tsutsumi, H., Hamachi, H., Tsumoto, K., Molecular Basis of Recognition of Antibacterial Porphyrins by Heme-Transporter IsdH-NEAT3 of Staphylococcus aureus, Biochemistry, 10.1021/bi200493h, 50, 34, 7311-7320, 2011.08, [URL], 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..
50. Yanaka, S., Sano, E., Naruse, N., Miura, K.-I., Futatsumori-Sugai, M, Caaveiro, J.M.M., Tsumoto, K., Non-core Region Modulates Interleukin-11 Signaling Activity: Generation of agonist and antagonist variants, J. Biol. Chem., 10.1074/jbc.M110.152561, 286, 10, 8085-8093, 2011.03, [URL], 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..
51. Abe, R., Caaveiro, J.M.M., Kudo, M., Tsumoto, K., Solubilization of membrane proteins with novel N-acylamino acid detergents, Mol. Biosyst., 10.1039/b925791h, 6, 4, 677-679, 2010.04, [URL], 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..
52. Landon, M., Lieberman, R.L., Hoang, Q.Q., Shulin, J., Caaveiro, J.M.M., Orwig, S, Kozakov, D, Brenke, R., Chuang, G.-Y., Beglov, D., Vajda, S., Petsko, G.A., Dagmar, R., Detection of ligand binding hot spots on protein surfaces via fragment-based methods: application to DJ-1 and glucocerebrosidase, J. Comput. Aid. Mol. Des., 10.1007/s10822-009-9283-2, 23, 8, 491-500, 2009.08, [URL], 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..
53. Sigala, P.A., Caaveiro, J.M.M., Ringe, D., Petsko, G.A., Tsumoto, K., Hydrogen Bond Coupling in the Ketosteroid Isomerase Active Site, Biochemistry, 10.1021/bi900713j, 48, 29, 6932-6939, 2009.07, [URL], 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..
54. Sakamoto, S., Caaveiro, J.M.M., Sano, E., Tanaka, Y., Kudo, M., Tsumoto, K., Contributions of Interfacial Residues of Human Interleukin15 to the Specificity and Affinity for Its Private alpha-Receptor, J. Mol. Biol., 10.1016/j.jmb.2009.04.050, 389, 5, 880-894, 2009.06, [URL], 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..
55. Sigala, P.A., Kraut, D.A., Caaveiro, J.M.M., Pybus, B., Ruben, E.A., Ringe, D., Petsko, G.A., Herschlag, D., Testing geometrical discrimination within an enzyme active site: Constrained hydrogen bonding in the ketosteroid isomerase oxyanion hole, J. Am. Chem. Soc., 10.1021/ja803928m, 130, 41, 13696-13708, 2008.10, [URL], 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..
56. Barlic, A., Gutierrez-Aguirre, I., Caaveiro, J.M.M., Cruz, A., Ruiz-Arguello, M.B., Perez-Gil, J., Gonzalez-Manas, J.M., Lipid phase coexistence favors membrane insertion of equinatoxin-II, a pore-forming toxin from Actinia equina, J. Biol. Chem., 10.1074/jbc.M313817200, 279, 33, 34209-34216, 2004.08, [URL], 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..
57. Xie, Y.C., Das, P.K., Caaveiro, J.M.M., Klibanov, A.M., Unexpectedly enhanced stereoselectivity of peroxidase-catalyzed sulfoxidation in branched alcohols, Biotechnol. Bioeng., 10.1002/bit.10308, 79, 1, 105-111, 2002.07, [URL], 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..
58. Das, P.K., Caaveiro, J.M.M., Luque, S., Klibanov, A.M., Asymmetric sulfoxidations mediated by alpha-chymotrypsin, Biotechnol. Bioeng., 10.1002/bit.10187, 78, 1, 104-109, 2002.04, [URL], 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..
59. Das, P.K., Caaveiro, J.M.M., Luque, S., Klibanov, A.M., Binding of hydrophobic hydroxamic acids enhances peroxidase's stereoselectivity in nonaqueous sulfoxidations, J. Am. Chem. Soc., 10.1021/ja012075o, 124, 5, 782-787, 2002.02, [URL], 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..
60. Caaveiro, J.M.M., Echabe, I., Gutierrez-Aguirre, I., Nieva, J.L., Arrondo, J.L., Gonzalez-Manas, J.M., Differential interaction of equinatoxin II with model membranes in response to lipid composition, Biophys. J., 80, 3, 1343-1353, 2001.03, [URL], 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..
61. Caaveiro, J.M.M., Gutierrez-Aguirre, I., Tribout, M., Paredes, S., Gonzalez-Manas, J.M., An evaluation of some model systems commonly used in the study of the sea anemone toxin, Acta Biol. Slov., 43, 1-2, 93-97, 2000.06, [URL], 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..
62. Caaveiro, J.M.M., Molina, A., Rodriguez-Palenzuela, P., Goni, F.M., Gonzalez-Manas, J.M., Interaction of wheat alpha-thionin with large unilamellar vesicles, Protein Sci., 7, 12, 2567-2577, 1998.12, [URL], 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..
63. Caaveiro, J.M.M., Molina, A., Gonzalez-Manas, J.M., Rodriguez-Palenzuela, P., Garcia-Olmedo, F., Goni, F.M., Differential effects of five types of antipathogenic plant peptides on model membranes, FEBS Lett., 410, 2-3, 338-342, 1997.06, [URL], 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..
64. Taneva, S.G., Caaveiro, J.M.M., Muga, A., Goni, F.M., A pathway for the thermal destabilization of bacteriorhodopsin, FEBS Lett., 367, 3, 297-300, 1995.07, [URL], 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..
65. Taneva, S.G., Caaveiro, J.M.M., Petkanchin, I.B., Goni, F.M., Electrokinetic charge of the anesthetic-induced BR(480) and BR(380) spectral forms of bacteriorhodopsin, Biochim. Biophys. Acta, 1236, 2, 331-337, 1995.06, [URL], 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..