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

Professor / Faculty of Pharmaceutical Sciences

1. K. Fujimoto, S. Nagatoishi, K. Tsumoto, T. Ueda, and J. Caaveiro, Biophysical screening in nanodiscs reveals novel inhibitors of the bacterial ABC transporter MsbA., The Protein Society, 2020.07, [URL], Development of new antibiotics is urgently needed to combat emerging resistance in bacteria. MsbA is an essential ABC-transporter located in the inner membrane of E. coli. MsbA is directly responsible for the export of Lipid A, the key precursor of lipopolysaccharide (LPS, also known as endotoxin). LPS is of critical importance for the bacterium survival, and therefore MsbA is a pharmacological target. Because of continuous improvements in biophysical methodologies, these techniques are increasingly employed for the screening of chemical compounds to protein targets. In this study, we sought to perform such screening in MsbA by surface plasmon resonance (SPR), a methodology that detects molecules binding to the target with great celerity. To enable the screening, we embedded MsbA in proteo-lipidic particles known as nanodiscs, which greatly stabilized the ATPase activity of the target. Despite the large difference in mass between the dimeric MsbA in nanodiscs and the average size of fragments (>1,000-fold) we detected robust binding of several compounds. The binding of these compounds was cross-validated by dose-dependence analysis and thermodynamic techniques. Moreover, the majority of these fragment compounds did not only bind to the target molecule, but also reduced the ATPase activity of MsbA with various intensities, from partial to complete inhibition. Our research has revealed the identity of novel compounds inhibiting the activity of an essential bacterial protein. We hope to develop these fragments into potent antibacterial compounds, and take the lessons learned to apply this approach to other protein targets of bacterial and human origin..
2. S., Valenciano-Bellido, Vu N.T., Nakakido, M., Caaveiro, J.M.M, and Tsumoto, K.., Molecular Basis for Heme Extraction of the Antimicrobial Target IsdH from Staphylococcus aureus from Human Hemoglobin, Biophysical Society, 2020.02, [URL], Staphylococcus aureus is a pathogenic bacterium responsible for a majority of deadly hospital-acquired infections worldwide, and increasingly resistant to a broad range of antibiotics. This dangerous combination demands action to obtain more efficient antimicrobial approaches. The iron surface determinant (Isd) system comprises a group of S. aureus proteins that obtain iron from the host organism. Since iron is essential for bacterial survival and proliferation during infection, Isd has been proposed as an antibacterial target. In particular, the protein IsdH is exposed on the surface of the bacteria and able to sequester hemoglobin to acquire its heme molecule which contains an atom of iron. IsdH comprises three near-iron transporter (NEAT) domains binding hemoglobin (NEAT1 and NEAT2) and heme (NEAT3) connected by characteristic linker domains of unclear function. The objective of this study is to deciphering the molecular mechanism of heme extraction, including the role of the linker re- gion. The first evidence of the structure of IsdH linker-NEAT3 at high resolu- tion with heme bound is presented. In combination with thermodynamic and structural data we propose a extraction mechanism of heme by IsdH. Although the linker neither accelerate nor strengthen the binding of heme, it contributes to stabilize the critical NEAT3 domain necessary to capture heme. This research contributes to clarify the molecular basis for heme extraction by a po- tential antimicrobial target..
3. J.L. Nieva, E. Rujas, S. Insausti, D. P. Leaman, P. Carravilla, I. Iloro, R. Sánchez-Eugenia, L. Zhang, M. García-Porras, F. Elortza, C. Eggeling, J.-P. Julien, A. Ojida, M. B. Zwick, J. Caaveiro, Strengthening Interactions with the Membrane Interface through Grafted Aromatic Compounds produces extremely potent HIV-1 neutralizing antibodies, Biophysical Society, 2020.02, [URL], Broadly-neutralizing antibodies (bnAbs) against the Env glycoprotein guide the rational development of vaccines and therapeutic agents to prevent or treat HIV- 1 infection. In addition, bnAb-derived binding fragments are instrumental for the study of the structure-function relationships of the conformationally dynamic Env complex. Here, we demonstrate that the anti-viral activity of bnAbs against the Membrane-Proximal External Region of the Env glycoprotein can be enhanced via single-site modification of membrane-interacting Ab areas with synthetic aromatic compounds. Potency enhancement in cell-entry inhibition and standard neutralization assays correlated with an increase in affinity for the native antigen in virions and did not compromise neutralization breadth. Thus, we have established an opti- mization procedure with the potential of improving functionality of Abs that bind immunotherapeutic targets at membrane surfaces..
4. S. Insausti, E. Rujas, D. P. Leaman, P. Carravilla, I. Iloro, R. Sánchez-Eugenia, L. Zhang, M. García-Porras, F. Elortza, C. Eggeling, J.-P. Julien, A. Ojida, M. B. Zwick, J. Caaveiro, and J. L. Nieva, Potency enhancement of HIV-1 neutralizing antibodies by site-specific chemical modification., The Antibody Society / 2019 Antibody Engineering & Therapeutics, 2019.12, [URL], Engineered variants of broadly-neutralizing antibodies (bnAbs) have shown efficacy in passive immunotherapy of HIV-1 infection. Here, we demonstrate that the anti-viral activity of bnAbs against the Membrane-Proximal External Region of the HIV-1 Env glycoprotein can be enhanced up to 103-fold, via single-site modification of membrane-interacting areas with linear and polycyclic aromatic chemical compounds. In an extreme case, chemical modification of a distant site complements ablation of the heavy-chain complementarity determining region 3 (HCDR3) loop apex, resulting in the functional recovery of a fully inactive antibody. Thus, we have established a procedure based on the use of synthetic molecules to improve functionality of antibodies against HIV. .
5. J.L. Nieva, E. Rujas, S. Insausti, R. Sánchez-Eugenia, M. García-Porras, B. Jiménez-Díaz, V. Monceaux, J.-P. Julien, I. Angulo-Barturen, A. Saéz-Cirión, A. Ojida, and J. Caaveiro, Grafting synthetic aromatic compounds improves the potency of a pan-neutralizing HIV antibody, PEGS, 2019.11, Engineered variants of broadly-neutralizing antibodies (bnAbs) have shown efficacy in passive immunotherapy of HIV-1 infection. Particularly, bnAbs raised against the neutralizing MPER epitope have gathered much attention in the area because they can potentially block infection by the large variety of circulating HIV-1 strains and isolates. Despite their remarkable breadth, the potential clinical translation of Abs belonging to this class is hampered by their overall modest potency. Here, we employ cell-entry inhibition and ex-vivo infection neutralization assays to demonstrate that the potency of the MPER bnAb 10E8, can be enhanced up to 102-fold, via single-site modification with linear and polycyclic aromatic chemical compounds. That is, chemically modified 10E8 versions display potency levels comparable to those of bnAbs currently under clinical development. Thus, we have established a procedure based on the use of synthetic molecules to improve functionality of pan-neutralizing HIV antibodies..
6. S. Valenciao-Bellido, J. Caaveiro, K. Tsumoto, IsdH from Staphylococcus aureus: mechanism of action and novel antibacterial strategies, Chem-Bio Informatics Society, 2019.10, [URL], The bacterium Staphylococcus aureus is the major cause of deadly hospital-acquired infections worldwide1. The Isd (Iron surface determinant) system is a group of proteins that obtain iron from the host organism2,3, helping the bacterium to proliferate, and therefore a promising antibacterial target. The protein IsdH binds to hemoglobin and acquires the heme4,5. IsdH comprises three NEAT (Near-iron transporter) domains connected by linkers of unknown function6,7. The objectives of this study are deciphering the molecular mechanism of heme extraction, to explain the role of the linker region and to obtain an antibody that recognizes IsdH specifically while simultaneously inhibiting heme binding. The first evidence of the structure of IsdH linker-NEAT3 bound to heme was reported in this research. The heme extraction mechanism of IsdH has been deciphered and the role of the linker as key for heme extraction has been explained through structure analysis. Herein we also report a novel VHH that binds to IsdH linker-NEAT3. The crystal structure of the complex of VHH bound to -NEAT3 was determined at high-resolution, showing that the VHH specifically recognizes the heme binding pocket, and explaining why this antibody inhibits heme acquisition by IsdH..
7. I. Kaneda, S. Nagatoishi, D. Kuroda, M. Nakakido, J. Caaveiro, K. Tsumoto, Thermodynamic, kinetic and computational analyses of the recognition mechanism of a flexible protein antigen by an antibody, Chem-Bio Informatics Society, 2019.10, [URL], Many proteins have both rigid and flexible regions, and change their conformations upon ligand binding. With current methodologies of antibody generation, it is often difficult to rationally obtain specific antibodies toward flexible epitopes on antigens due to the lack of our knowledge on molecular recognition between antibodies and flexible antigens. In this study, we focused on the interaction between an antibody and an antigen binding to metal ions, to obtain quantitative relationships about antibody-antigen interactions where the antigen is highly flexible. As a model system, we used the Fab region of a monoclonal antibody 5E1 whose antigen is Sonic hedgehog (Shh) protein, which also binds to Ca2+ and Zn2+ ions. In a previous study, 5E1 Fab had exhibited high affinity toward Shh regardless of the presence or absence of the metal ions [1]. However, the details of the interactions and physicochemical properties remains unclear.
In this study, to reveal the binding mechanism of 5E1 Fab and Shh, thermodynamic and kinetic parameters of the interactions were experimentally evaluated, and we observed drastic changes of binding enthalpy and entropy depending on the existence of the metal ions. In addition, we analyzed and discussed energetic contributions of some interface residues based on physicochemical measurements and molecular dynamics simulations with the Ala mutants as well as the wild-type. In addition to the interaction analyses, we also experimentally and computationally assessed the thermal stability of Shh with or without the metal ions, showing the dependency of the metal ions on the thermal stability of the antigen.
A co-crystal structure of Shh and 5E1 Fab with the metal ions was already solved [1]. In this study, we also succeeded in obtaining a crystal structure of the complex without the metal ions, enabling the structure-based understanding of the interaction with or without the metal ions. Putting together, our results could be a guideline for rational design of antibodies that recognize flexible antigens.
8. J.L. Nieva, B. Apellaniz, Caaveiro, J.M.M., Caravilla, P, Garcia-Vesga, A., Insausti, S., Largo, E., Requejo-Isidro, J., Rujas, E., Sanchez-Eugenia, R., and Torralba, J. , Membrane accommodation surfaces modulate the biological function of anti-HIV antibodies through semi-specific interactions, Spanish Society of Biochemistry and Molecular Biology, 2019.07, [URL], Binding of antibodies (Abs) 4E10 or 10E8 to the envelope gly- coprotein (Env) of HIV-1 exerts one of the broadest neutralizing activities against this virus described to date. These Abs recognize the helical juncture that connects the membrane-proximal external region (MPER) and the transmembrane domain (TMD) present in the Env subunit gp41. Thus, gaining insight into the mechanism underlying MPER-mediated neutralization breadth can contribute to developing more effective vaccines and immunotherapeutic agents against HIV. Both, 4E10 and 10E8, exhibit unusual adaptations to attain specific, high-affinity binding to the helical MPER epitope presented on the surface of the viral membrane interface. We report here that manipulation through mutagenesis can adjust the 4E10 and 10E8 paratope surfaces to the viral membrane through semi-specific electrostatic interactions, and thereby modulate their biological activity..
9. R. Kawade, D. Kuroda, J. Caaveiro, H. Akiba, S. Okumura, T. Maruyuma, K. Entzminger, and K. Tsumoto, K., The effects of protonation of a phosphorylated amino acid on the molecular recognition: Comparative studies of generic proteins and an antibody., Protein Society, 2019.07, [URL], Since phosphorylation of an amino acid plays important roles in various cellular processes, how phos- phorylated amino acids are recognized has been widely discussed. However, many previous studies assumed that the protonation state of a phosphate group was (PO32) despite the fact that the phosphate group (pKa ~7) would exist as an equilibrium mixture of the unprotonated state (PO32-) and the singly protonated state (PO3H) in physiological condition. To analyze effects of a protonation state on protein dynamics, we performed MD simulations of 4 different proteinphosphorylated peptide complexes in vari- ous biological processes in which each peptide contained a phosphoserine residue in the unprotonated or singly protonated state. Our result showed that the (PO32-) was more preferable to (PO3H-) in the interactions due to the larger mobility of the phosphate group in the (PO32-) state. Furthermore, we also obtained a monoclonal antibody toward a phosphorylated peptide by phage display, and X-ray crystal- lography, thermodynamic analysis, mutagenesis, and MD simulations showed this antibody captures both protonation states equally well. Putting together, our results suggest that even a single protonation could have a large effect on molecular recognition of a phosphate group..
10. Caaveiro, J.M.M., Rujas, E., Tsumoto, K., and Nieva, J.L., Antibodies that insert into the lipid membrane to capture the antigen., Faculty of Pharmaceutical Sciences, 2019.03, The remarkable breadth of broadly neutralizing antibodies (bNAbs) against the transmembrane protein gp41 of HIV-1 makes this type of antibodies suitable model system for structure-guided vaccine design and immunotherapeutics against AIDS. In particular, bNAbs directed against the membrane proximal external region (MPER) of gp41 are considered a paradigm because of the mechanism of recognition of the antigen seem to involve neighboring lipids of the viral membrane. Indeed, the concrete role of these lipids for the biological function of these bNAbs is still a subject of controversy. In this presentation I will address this debate for two bNAbs: 4E10 and 10E8.
First, we compared the partitioning ability of the 4E10 antibody and several of its variants in synthetic membranes (liposomes). The antibody was mainly mutated at the region of the paratope surface in contact with the membrane interface. We employed a physical separation approach (vesicle flotation) and quantitative fluorescence measurements (spectroscopic titration) to evaluate the interaction with the membrane. Our energetic analysis suggested that antibody 4E10 behaves as a peripheral membrane protein.
Second, we analyzed the structure and binding activities of the 10E8 antibody in membrane and membrane-like environments. The X-ray crystal structure of the Fab-peptide complex in detergents revealed for the first time that the epitope of 10E8 comprises a continuous helix spanning the gp41 MPER/transmembrane domain junction. Overall, these findings support the idea that lipids are critical for the binding mechanism of these antibodies. We hope this research will help to envision affordable and efficient vaccines against HIV-1..
11. Caaveiro, J.M.M., Protein-based therapeutics, University of Malaysia, 2018.11, The discovery of novel medicines to tackle human diseases such as cancer or autoimmune disorders is a pressing challenge that requires urgent attention from the medical and scientific communities. The incorporation of proteins in the therapeutic arsenal is changing the perception of how medicines work. In particular, therapeutic antibodies and antibody-based molecules comprise a large fraction of the total substances approved by regulatory agencies to treat various diseases during the last decade. In this presentation, I will describe our efforts to understand the mechanisms by which antibodies recognize antigens. For example, the unprecedented role of membrane lipids in the recognition of the HIV-1 protein gp41 by broadly neutralizing antibodies of the IgG1 class will be described in this presentation. Another key element of antibody function is the cellular responses triggered by the binding of immune-complexes to Fc. Factors modulating the cytotoxic effect of antibodies such as glycosylation will be discussed in structural and energetic terms. Our research contributes to understand the fundamental basis of antibody-antigen and antibody-receptor interactions, directing ongoing efforts to design proteins with even better therapeutic outcomes..
12. Caaveiro, J.M.M., Reversible transformation of a metamorphic membrane protein, SickKids Research Institute, 2018.09.
13. Caaveiro, J.M.M., Drug discovery efforts at the Green-Pharma Research Center for System Drug Discovery @KU, NCATS - NIH, 2018.09.
14. Caaveiro, J.M.M., Integration of biophysical techniques for the screening of fragment libraries and lead optimization, NCATS- NIH, 2018.09, [URL].
15. Caaveiro, J.M.M., Molecular recognition of IgG-Fc by Fcgamma receptors, Antibodies & Complement: Effector Functions, Therapies & Technologies, 2018.06, [URL], The binding of antibodies of the IgG class to Fcgamma receptors triggers cell effector functions critical for the successful immune response. At the molecular level, the recognition of the Fc region of IgG involves an intricate network of non-covalent and weak interactions leading a broad range of affinities. As in drug binding to its biological target, or an inhibitor to an enzyme, changes in this carefully crafted three-dimensional arrangement of atoms leads to profound differences in the strength of protein-receptor partnerships. The small differences in the sequence of Fcgamma receptors lead to a broad range of binding responses with biological consequences. In this presentation the molecular mechanisms explaining the unique strength of the binding between IgG1-Fc and FcgammaRI (high-affinity receptor) will be analyzed from structural and thermodynamic points of view, including a discussion in depth about two antagonistic mechanisms proposed in recent times. In addition to geometric factors, it is increasingly evident that dynamic aspects of the binding partners also influence the interaction between IgG and Fcgamma receptors. In particular, the role of the glycan portion attached to both antibody and receptor is essential for the intensity of the biological response. By engineering a Fcgamma receptor, the role of the glycans is further clarified, suggesting that the composition of the glycan influence the degree of mobility (disorder) of the Fc portion of IgG. Overall, the accumulating evidence suggest that multifaceted interactions between IgG-Fc and Fcgamma receptors explain, at least in part, the immune effector function..
16. Nieva, J.L., Rujas, E., Insausti, S., Leaman, D.P., Apellaniz, B., Torralba, J., Zhang, L., Caaveiro, J.M.M., and Zwick, M.B. , Molecular recognition at the membrane interface: protein-membrane electrostatic interactions modulate the biological function of anti-HIV antibodies, Biophysical Society, 2018.02, [URL], Binding of antibodies 4E10 or 10E8 to the helical juncture that connects the membrane-proximal external region (MPER) and the transmembrane domain (TMD) of the Envelope glycoprotein (Env) gp41 subunit, translates into one of the broadest neutralizing activities against HIV-1 described to date. Accordingly, these antibodies and their mechanisms of action provide timely models in the development of effective vaccination and immunotherapeutic strategies. Both, 4E10 and 10E8, exhibit unusual adaptations to attain specific, high-affinity binding to the helical MPER epitope anchored into the viral membrane interface. We report here that manipulation of the paratope surface through mutagenesis can be used to adjust the electrostatic interactions of 4E10 and 10E8 with the membrane, and modulate their biological activity (neutralization potency and polyreactivity)..
17. Caaveiro, J.M.M., The Fascinating journey of a metamorphic protein to the cell membrane, Bioforo 5th Alumni Research Meeting, 2017.12, Dr. Caaveiro’s research program combines structural and thermodynamic data to address a broad range of biologically inspired phenomena. His ultimately goal is to understand the physicochemical basis underpinning biomolecular recognition at the atomic scale. Recently, Dr. Caaveiro and co-workers has revealed an unprecedented role for lipids in the pore-forming mechanism of the potent hemolytic class of actinoporins..
18. Tsumoto, K. and Caaveiro, J.M.M.* (*) presenter, Protein-lipid interactions in a pore-forming toxin., 55th Annual Meeting of the Biophysical Society of Japan, 2017.09, [URL], Pore-forming toxins (PFTs) are metamorphic proteins that fold stably in water solutions as well as in cellular membranes, where they assemble to form lethal transmembrane holes producing cell death. Actinoporins are a family of potent hemolytic toxins vigorously studied as a paradigm of alpha-helical PFTs in the context of lipid–protein interactions. We have revealed for the first time the complete energetic and structural basis of the transformation of a PFT from the water-soluble to the active transmembrane state, and its reverse process. We will show that FraC, an actinoporin, undergoes a stepwise metamorphosis towards the lethal pore that is catalyzed by the physicochemical properties of the membrane and by key non-covalent interactions with specific lipids..
19. Caaveiro, J.M.M., Molecular recognition phenomena involving proteins of therapeutic interest., 3rd HU-TMU-KU Joint Symposium for Pharmaceutical Sciences, 2017.09, The vast majority of biochemical processes in the cell involve, at some level, an intimate contact between the participating molecules. Proteins are key contributors in many molecular recognition events by directly interacting with other protein partners or with some other biomolecules such as DNA, lipids or carbohydrates. These recognition phenomena are based on carefully orchestrated non-covalent interactions comprising atoms from the protein, the partner molecule, and also from the solvent.
Our experimental design employs a mixture of high-resolution techniques including X-ray crystallography, titration and scanning calorimetry, and surface plasmon resonance. We obtain a detailed structural and energetic understanding of recognition phenomena in vitro, whose implications are evaluated by complementary biological and biochemical assays.
In this presentation, I will briefly summarize our contributions toward the clarification of several interaction phenomena involving proteins of therapeutic interest, such as the mechanism of action of certain anti-HIV-1 broadly-neutralizing antibodies, the recognition of antibodies by cellular receptors, the lipid-mediated metamorphosis of pore forming proteins, and the discovery and optimization of the first generation of inhibitors for the Parkinson’s disease associated protein DJ-1 and the antimicrobial target CapF. On the basis of this presentation, we are actively seeking novel directions in our research through collaboration with other fellow scientists..
20. Nieva, J.L., Rujas, E., Caaveiro, J.M.M., Insausti, S., Apellaniz, B., Jimenez, M.A. and Julien, J.P. , Structural basis of HIV entry and its inhibition by neutralizing antibodies against the fusogenic glycoprotein subunit 41, Spanish Society for Virology, 2017.06.
21. Caaveiro, J.M.M., Thermodynamic properties of the transition state in biomolecular recognition phenomena., 23rd Pharma-Science Forum, 2017.03, A complete description of the molecular and energetic factors present in biomolecular recognition phenomena mediated by proteins will facilitate the rational design of more advanced therapeutic approaches involving antibodies (and their derivatives). Numerous studies have described, in great detail, the structural, kinetic, and thermodynamic properties in the interaction between antibodies and their antigens. However, far less is known about the initial contacts and subsequent transition state leading to the formation of stable antibody-antigen complexes. By using the Eyring formalism, we have addressed this problem from a thermodynamic perspective using two different antibody-antigen systems. First, we describe recognition of MCP-1 (monocyte chemotactic protein-1), an important therapeutic target in inflammatory diseases, by the mature antibody 11K2. Mutagenesis analysis, inspired by computational calculations, demonstrated that basic thermodynamic parameters in the transition state are correlated with the improvement of affinity. Secondly, we examined a special group of antibodies vigorously studied as anti-infectious agents in the fight against AIDS. The broadly neutralizing antibody 4E10 recognizes the MPER (membrane-proximal external region) epitope in a very different environment, the membrane interface, still the thermodynamic signature at the transition state is similar to that of 11K2. The generality of our observations was evaluated independently in a different model system. We conclude that improving intermolecular interactions in the transition state may translate in higher affinity and durability of the binding complexes..
22. Caaveiro, J.M.M., Biomolecular recognition: a structural and physicochemical perspective., Chile-Japan Academic Forum 2016 at Patagonia, 2016.11.
23. Caaveiro, J.M.M., Thermodynamic tools in fragment-based drug discovery., 2016 UT-PUCC Joint Workshop for Frontiers in Bioengineering and Biomedical Engineering, 2016.11.
24. Caaveiro, J.M.M., Pore formation assisted by lipids., Membrane pores: from structure and assembly, to medicine and technology, 2016.06, [URL], Pore-forming toxins (PFT) constitute a fascinating group of proteins belonging to the molecular offensive and defensive machinery of virtually all kingdoms of life. This class of water-soluble proteins shares the remarkable ability to metamorphose in the presence of cell membranes, generating lytic pores and causing cell-damage. Actinoporins are a family of potent hemolytic toxins from sea anemone forming alpha-helical pores on cellular and model membranes. In general, two requirements are sufficient to trigger pore-formation by actinoporins: (i) the presence of the lipid sphingomyelin, and (ii) the segregation of the membrane on domains or lipid-rafts. Until recently, the molecular basis of pore-formation by actinoporins, and specially the specific requirement for sphingomyelin were unclear. However, a number of recent studies have shed light into critical steps of their mechanism of action, such as binding of the toxins to the membrane, self-assembly via protein-protein interactions, and assembly of the transmembrane pore. Collectively, the data suggests that sphingomyelin facilitates pore-formation at the binding and assembly stages, and reveal the first example of a hybrid lipid/protein pore by a PFT. The structural and thermodynamic basis of this novel architecture will be explained in detail during this presentation. Surprisingly, the entire process can be made reversible under mild experimental conditions by the careful selection of detergents, challenging current perceptions in the field of membrane-protein interactions..
25. Caaveiro, J.M.M., Tanaka, K., Morante, K., and Tsumoto, K., Basis of cell-membrane damage by a protein nanopore., Todai-Tsinghua Joint Workshop for Frontiers in Bioengineering and Biomedical Engineering, 2016.03, Pore-forming toxins (PFT) are water-soluble proteins that possess the remarkable ability to self-assemble on the membrane of target cells, where they form pores causing cell damage. We have elucidated the mechanism of action of the haemolytic PFT fragaceatoxin C (FraC), by determining crystal structures at different stages of the lytic mechanism, namely the water-soluble state, the monomeric lipid-bound form, an assembly intermediate and the fully assembled transmembrane pore. The structure of the transmembrane pore exhibits a unique architecture composed of both protein and lipids, with some of the lipids lining the pore wall, acting as assembly cofactors. The pore also exhibits lateral fenestrations that expose the hydrophobic core of the membrane to the aqueous environment. The incorporation of lipids from the target membrane within the structure of the pore provides a membrane-specific trigger for the activation of a haemolytic toxin. In addition, we describe the use of detergents with different physicochemical properties to achieve the spontaneous conversion of transmembrane pores of FraC back into the initial water-soluble state, challenging the Anfinsen dogma. Thermodynamic and kinetic stability data suggest that specific detergents cause an asymmetric change in the energy landscape of the protein, allowing the bidirectional transformation of a membrane protein. Our studies also have unveiled promising candidates for the development of protein-based biosensors highly sensitive to the concentration of cholesterol in biological membranes..
26. Caaveiro J.M.M., Miyafusa, T., Chigira, T., Nakano, K., Nagatoishi, S., and Tsumoto, K., Structural and mechanistic basis of capsular polysaccharide-synthesizing enzymes CapE/F, and the route to discovery novel inhibitors with antibacterial properties., 7th International Chemical Congress of Pacific Basin Societies PacificChem, 2015.12, [URL], The capsular polysaccharide (CP) forms a mucous layer on the outer surface of pathogenic Staphylococcus aureus critical for bacterial resistance. Because of the key role of CP in pathogenesis, the discovery of inhibitors targeting enzymes of the biosynthetic pathway of CP may lead to new classes of antibacterial compounds. But before getting there, it is necessary to gain a fundamental understanding of the structure, function, and mechanism of these enzymes. In this presentation I will discuss the structure/function/energetics of the coupled enzymes CapE and CapF that, together with CapG, are responsible for the synthesis of the CP precursor UDP-L-FucNAc. CapE belongs to a new subfamily of SDR enzymes present only in pathogenic strains of Gram-positive bacteria. CapE is characterized by a unique catalytic machinery and a highly dynamic hexameric organization [1,2]. Remarkably CapE undergoes large conformational changes in each catalytic cycle involving ~30% of its residues and also the cofactor NADP+. These conformational changes seem critical to generate the kinetic product instead of the more stable thermodynamic product. The enzyme CapF belongs to a new class of bifunctional enzyme composed of a metallo-cupin domain fused to a SDR domain [3]. The new crystal structure of CapF and the mechanistic data revealed the order and location of the chemical reactions occurring in each domain. We also identified unambiguously the metal ion in the cupin domain as Zn2+. The metal ion has crucial roles not only in catalysis, but also in the overall stability of the protein. Finally, I will describe our ongoing efforts to generate novel antibacterial inhibitors using fragment-based methodologies [4].
[1]Miyafusa et al. (2013) Biosci. Rep. 33:463-474, DOI: 10.1042/BSR20130017
[2]Miyafusa et al. (2013) FEBS Lett. 587:3824-3830, DOI: 10.1016/j.febslet.2013.10.009
[3]Miyafusa et al. (2011) Biochem. J. 443:671-670, DOI: 10.1042/BJ20112049
[4]Nakano et al., unpublished observations.
27. Caaveiro J.M.M., Kobe, A., Mitani, T., and Tsumoto, K., Use of thermodynamic tools in the early stages of fragment-based drug discovery., 7th International Chemical Congress of Pacific Basin Societies PacificChem, 2015.12, [URL], Fragment-based drug discovery (FBDD) has enjoyed increasing popularity in academia during the screening stage of numerous drug discovery campaigns. However, the small size of the chemical compounds used in FBDD requires not only more sensitive instruments, but also novel approaches to extract quality data using fewer resources. In this presentation I will introduce SITE (single-injection thermal extinction), a thermodynamic methodology based on a modification of the well-known technique of isothermal-titration calorimetry (ITC). SITE is a fast calorimetric competitive assay suitable for automation that captures the essence of ITC but using significantly less time and fewer resources. In this presentation I will describe the principles and advantages of this methodology with three successful examples, the enzymes ketosteroid isomerase, MAP kinase ERK2, and carbonic anhydrase II. The data obtained with SITE is also evaluated versus a panel of other screening methodologies based on biophysical principles. We propose SITE as a useful tool in the identification of high-quality hits with high enthalpic efficiency in the early stages of drug discovery, and in particular that of FBDD.

Kobe et al. (2013) J. Med. Chem. 56:2155-2159, DOI: 10.1021/jm301603n.
28. Caaveiro, J.M.M. , Principles of biomolecular recognition of IgG-Fc by its receptors., 2nd FcR Research Forum, 2015.10, [URL].
29. Caaveiro J.M.M., Kobe, A., Tashiro, S., Kajihara, D., Kikkawa, M., Mitani T., and Tsumoto K., Thermodynamic Tools in the Early Stages of Drug Discovery., The 4th Asia-Pacific Protein Association (APPA) Conference, 2014.05, [URL], There is an increasing need to develop more precise methods in the process of drug discovery. In this regard, fragment-based drug discovery (FBDD) has enjoyed increasing popularity as screening method in the early stages of drug discovery. The small size of the fragments used in FBDD requires not only more sensitive instruments, but also novel approaches to extract quality data using fewer resources. In this presentation I will introduce SITE (single-injection thermal extinction), a thermodynamic methodology that selects high-quality hits early in FBDD [1]. SITE is a fast calorimetric competitive assay suitable for automation that captures the essence of isothermal titration calorimetry but using significantly less time and materials. In this presentation I will describe the principles and advantages of this methodology with practical examples..
30. Caaveiro, J.M.M., Thermodynamic tools in the early stages of drug discovery., 6th GCOE Medical Sciences Mini-symposium, 2013.02.
31. Caaveiro, J.M.M. and Tsumoto, K., Expanding the horizons of isothermal titration calorimetry, The Japan Society for Calorimetry and Thermal Analysis, 2012.09.
32. Caaveiro, J.M.M., Sources of specificity in weak and ultraweak biomolecular interactions., Alumni association of the Institute of Medical Science, The University of Tokyo, 2011.12, Proper functioning of biological systems demands exquisite recognition of binding partners at the molecular level. How to achieve such high specificity in the complex environment of the cell? Intensive research in this field has revealed the physicochemical nature of many of the forces involved in recognition phenomena, such as in antigen-antibody, ligand-receptor, or enzyme-substrate complexes. This detailed knowledge has facilitated the design of novel and better drugs to combat human disease. However, there remains a large pool of recalcitrant “transient” associations that are still poorly understood. Our next grand challenge consists in the detailed mapping, cataloging, and biophysical characterization of weak and ultraweak biomolecular interactions. These investigations are expanding at a rapid pace thanks to technological and conceptual innovations in multiple fronts. In this presentation, I will discuss two biological systems displaying distinctive weak and/or ultraweak binding properties: (1) lipid-protein, and (2) protein-sugar interactions. In the first example I will review mechanisms by which lipid composition may modulate the optimal activity of membrane proteins and lytic peptides. In addition, I will discuss how pore-forming proteins may exploit small physical imperfections on the surface of the membrane to induce cell lysis. In the second example, I will introduce a sugar-processing enzyme (GalM) as a model system to study ultraweak protein-sugar interactions. In this study we have deepened our understanding about the nature of C-Hπ interactions in modulating sugar recognition and binding affinity. Overall, our data supports an increasingly important role of aromatic interactions and water-mediated contacts during transient biomolecular associations. Future challenges will be discussed if time permits..
33. Caaveiro, J.M.M., Moriwaki, Y, Abe, R., Watanabe, M., Tanaka, Y., Vu, N., and Tsumoto, K., Thermodynamic analysis of binding and transfer of heme: Isd system., 14th Asian Chemistry Congress, 2011.09, The emergence and proliferation of bacteria carrying antibiotic resistance is a serious problem that threatens public health. This is particularly grave in certain multi-resistant strains of bacterium Staphylococcus aureus (so-called superbug). In order to sustain effective infection, this bacterium steals the iron atom contained in the heme group of hemoglobin from the host organism. The heme acquisition system of S. aureus is composed by more than ten proteins collectively known as Iron-regulated surface determinant (Isd). Isd proteins are strategically located all the way from the cell surface to the interior of the cell. Heme molecules are transferred among Isd proteins in an orderly and sequential fashion. Blocking this critical iron supply-route in S. aureus is an attractive therapeutic possibility that may alleviate disease and suffering. However, we must investigate the molecular basis of heme transfer within Isd before this therapeutic approach can be brought to fruition. We are particularly interested in understanding (i) the extent of binding of heme to Isd proteins, (ii) the nature of protein-protein interactions among Isd members, and (iii) the thermodynamic and structural factors underlying these complex interactions. We brought together isothermal titration calorimetry, UV-visible spectrometry, circular dichroism, site-specific mutagenesis, and high-resolution X-ray crystallography to understand the energetic and dynamic properties controlling the first stages of heme acquisition. We found out that the initial stages in the internalization route require binding of heme ligand to receptors with high affinity. We also observed that transfer of heme towards the interior of the cell is fast and largely irreversible, thus avoiding back-transfer reaction and limiting the loss of precious heme molecules back to solution. Our results also suggested that heme transfer is facilitated by specific protein-protein interactions between sequential Isd receptors. Moreover, by using non-iron metalloporphyrins (i.e. the equivalent of heme but containing a metal other than iron) we demonstrated that electrostatic forces mediate recognition of ligand. X-ray crystal structures of receptor IsdH with heme and other non-iron porphyrins clarified some of the energetic attributes determined in our thermodynamic analyses. We hope our study will help to develop new therapeutic agents against S. aureus. .
34. Caaveiro, J.M.M., Kobe, A., Clifton, J., Ringe, D., Petsko, G.A., and Tsumoto, K., Aromatic interactions are essential for the binding of galactose to a sugar processing enzyme., 6th International Chemical Congress of Pacific Basin Societies PacificChem, 2010.12, Protein-carbohydrate interactions regulate numerous molecular recognition events, such as bacterial and viral infections, cell adhesion in inflammation and metastasis, and many other intracellular and signal transduction pathways. Although numerous crystals structures of carbohydrate binding proteins are available in the Protein Data Bank, there remain conflicting views about the exact nature of the forces involved in carbohydrate binding and recognition. In this study we aimed at understanding the nature of the forces involved in the binding of galactose glucose to the model enzyme Galactose Mutarotase (GalM) from Escherichia coli. First, the crystal structure of GalM with Galactose bound was solved by the Multiwavelength Anomalous Diffraction (MAD) method, and the phases extended to a final resolution of 1.4 Å. E. coli GalM forms a β-sandwich that closely resembles that of the homolog enzyme from Lactobacillus lactis [1]. The active site displays numerous polar residues that orient the substrate towards catalytic residues Glu309, His104 and His175. Substitution of these residues did not significantly alter the architecture of the binding site, while dramatically diminished the enzymatic activity. Aromatic residue Tyr284 is located at the bottom of the binding site towards the alpha-face of the galactose moiety. High-resolution X-ray crystallography, Isothermal Titration Calorimetry, and enzymatic assays performed with wild type and Tyr284-substituted proteins revealed that the presence of an aromatic residue at this position is essential for binding of the sugar moiety, although the substitutions do not completely abolish the activity of the enzyme. More importantly, this effect is exclusively of aromatic nature, since hydrophobic residues at that position do not promote binding of the sugar.
35. Caaveiro, J.M.M. , Enzymology in silico., University of the Basque Conuntry, 2002.01.
36. Caaveiro, J.M.M., Enzymology in silico, University of Zaragoza, 2002.01.
37. Caaveiro, J.M.M., Forum-Iberia for the promotion of science and knowledge in Spain, National Biotechnology Center (CSIC), 2001.12.
38. Caaveiro, J.M.M., Gutiérrez-Aguirre, I., Tribout, M., Paredes, S., and González-Mañas, J.M., An evaluation of some model systems commonly used in the study of the interaction of the sea anemone toxin equinatoxin II with membranes., 3rd Meeting of the Slovenian Biochemical Society, 2001.09, [URL].
39. Caaveiro, J.M.M., Aguirre, I. G., Goñi, F. M., and González-Mañas, J.M., Interaction of alpha-thionin with lipidic vesicles, Workshop on Eukaryotic Antibiotic Peptides (Organized by the Institute Juan March), 1999.02.