Role of Tyr Residues in the Contact Region of Anti-lysozyme Monoclonal Antibody HyHEL10 for Antigen Binding

Tsumoto, Kouhei; Ogasahara, Kyoko; Ueda, Yoshitaka; Watanabe, Kimitsuna; Yutani, Katsuhide; Koyanagi, Izumi

doi:10.1074/jbc.270.31.18551

Cited by 68 publications

(51 citation statements)

References 37 publications

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“…Although dimer formation via disulfide bonds was observed in each deletion mutant, replacement of the sole Cys residue with Ser had no effect on either oligomerization or thermostability of the protein (data not shown), suggesting that the disulfide bond between these Cys residues also makes little contribution to the thermostability of the protein. Taken together, these results suggest that adequate association of monomers involving the major contact region is critical for hexamerization and hyperthermostability and also suggest that the three residues, ligand interactions (51,52), antigen-antibody interactions (53,54), and homodimer formation (55,56), we propose that these residues also be defined as hot spot residues (57) for hexamerization and/or hyperthermostability.…”

Section: Discussionmentioning

confidence: 97%

How Oligomerization Contributes to the Thermostability of an Archaeon Protein

Tanaka

Tsumoto

Yasutake

et al. 2004

Journal of Biological Chemistry

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To study how oligomerization may contribute to the thermostability of archaeon proteins, we focused on a hexameric protein, protein L-isoaspartyl-O-methyltransferase from Sulfolobus tokodaii (StoPIMT). The crystal structure shows that StoPIMT has a distinctive hexameric structure composed of monomers consisting of two domains: an S-adenosylmethionine-dependent methyltransferase fold domain and a C-terminal ␣-helical domain. The hexameric structure includes three interfacial contact regions: major, minor, and coiled-coil. Several C-terminal deletion mutants were constructed and characterized. The hexameric structure and thermostability were retained when the C-terminal ␣-helical domain (Tyr 206 -Thr 231 ) was deleted, suggesting that oligomerization via coiled-coil association using the C-terminal ␣-helical domains did not contribute critically to hexamerization or to the increased thermostability of the protein. Deletion of three additional residues located in the major contact region, Tyr 203 -Asp 204 -Asp 205 , led to a significant decrease in hexamer stability and chemico/thermostability. Although replacement of Thr 146 and Asp 204 , which form two hydrogen bonds in the interface in the major contact region, with Ala did not affect hexamer formation, these mutations led to a significant decrease in thermostability, suggesting that two residues in the major contact region make significant contributions to the increase in stability of the protein via hexamerization. These results suggest that cooperative hexamerization occurs via interactions of "hot spot" residues and that a couple of interfacial hot spot residues are responsible for enhancing thermostability via oligomerization.Thermophilic organisms grow optimally at temperatures of Ͼ70°C. The proteins isolated from these organisms are usually more thermostable than homologous proteins from mesophiles, despite the fact that they have similar three-dimensional structures and identical catalytic mechanisms to the mesophilic proteins as well as sequence homologies of 40 -85% (1). Extensive studies have focused on identifying key determinants or common factors responsible for the thermostability of thermophile proteins, and the following structural features have been proposed as responsible for the high thermostability: decreased solvent-exposed surface area (2), increased polar interactions at the molecular surface (3-10), higher packing density within the protein (10 -12), greater core hydrophobicity (10, 13-15), shorter surface loops (11, 16), and more hydrogen bonds (7,10,(17)(18)(19) relative to those of ordinary proteins as well as oligomerization (10, 20 -25).The protein L-isoaspartyl-O-methyltransferase (PIMT 1 ; EC 2.1.1.77) functions as an enzyme in the repair of age-damaged proteins in which asparagines and aspartates have been spontaneously deamidized and isomerized into L-isoaspartyl residues; it catalyzes S-adenosylmethionine (AdoMet)-dependent methylation of the ␣-carboxyl group of the L-isoaspartyl residues to form L-iso-Asp-␣-methyl ester (26,27...

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Section: Discussionmentioning

confidence: 97%

How Oligomerization Contributes to the Thermostability of an Archaeon Protein

Tanaka

Tsumoto

Yasutake

et al. 2004

Journal of Biological Chemistry

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“…The quantitative contributions of Asnl9, Arg21, AsplOl, and Gly102 were evaluated earlier by sitedirected mutagenesis (Kam-Morgan et al, 1993). Complementary site-directed mutagenesis investigations of some of the HyHEL-IO Fv paratope residues have been published (Tsumoto et al, 1995(Tsumoto et al, , 1996. The present work was undertaken to extend the previous investigations by systematically probing the roles of the remaining amino-acid residues in the discontinuous epitope for HyHEL-IO.…”

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confidence: 94%

Quantitative evaluation of the chicken lysozyme epitope in the HyHEL‐10 fab complex: Free energies and kinetics

1998

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The hen (chicken) egg-white lysozyme (HEWL) epitope for the monoclonal antibody HyHEL-IO Fab (Fab-IO) was investigated by alanine scan mutagenesis. The association rate constants (/con) for the HEWL.Fab-10 complexes were obtained from the homogenous solution method described in the preceding paper (Taylor et al., 1998). A new method for determining the dissociation rate constant (kOff) for the complex, by trapping nascent free antibody with an inactive HEWL mutant is described. The values of k,, fall within a factor of 2 of the wild-type (WT) HEWL value (1.43 f 0.13 X lo6 M" s-'), while the increases in kc,w more nearly reflect the total change in free energies of the complex (AAG"). The dissociation constants (KO) were measured directly in those cases where satisfactory kinetic data could not be obtained. The Y20A, K96A, and K97A HEWL.Fab-IO complexes are destabilized by more than 4 kcal/mol compared to the WT complex. The R21A, L75A, and DlOlA antibody complexes are moderately destabilized (0.7 < AAGo 5 1 .O kcal/mol). Additional mutations of the "hotspot" residues (TYRO, Lys96, Lys97) were constructed to probe, more precisely, the nature of their contributions to complex formation. The results show that the entire hydrocarbon side chains of Tyr20 and Lys97, and only the s-amino group of Lys96, contribute to the stability of the complex. The value of AAG,] for the R21A mutant complex is a distinct outlier in the Arg2l replacement series demonstrating the importance of supplementing alanine scan mutagenesis with additional mutations.

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“…In many antibodies, Tyr residues located at the surface are critical for antigen binding (45)(46)(47)(48)(49)(50)(51)(52)(53), and Tyr residues often have an important role in other protein-protein interactions (45,46,54,55). In the following sections, we focus on the role of an aromatic ring to the polyether-antibody interaction, which was revealed by systematic mutation of L-Tyr-91.…”

Section: Role Of Each Residue In the Interaction Of Ctx3c-abc With Thmentioning

confidence: 99%

Critical Contribution of Aromatic Rings to Specific Recognition of Polyether Rings

Tsumoto

Yokota

Tanaka

et al. 2008

Journal of Biological Chemistry

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To address how proteins recognize polyether toxin compounds, we focused on the interaction between the ABC ring compound of ciguatoxin 3C and its specific antibody, 1C49. Surface plasmon resonance analyses indicated that Escherichia coliexpressed variable domain fragments (Fv) of 1C49 had the high affinity constants and slow dissociation constants typical of antigen-antibody interactions. Linear van't Hoff analyses suggested that the interaction is enthalpy-driven. We resolved the crystal structure of 1C49 Fv bound to ABC ring compound of ciguatoxin 3C at a resolution of 1.7 Å . The binding pocket of the antibody had many aromatic rings and bound the antigen by shape complementarity typical of hapten-antibody interactions. Three hydrogen bonds and many van der Waals interactions were present. We mutated several residues of the antibody to Ala, and we used surface plasmon resonance to analyze the interactions between the mutated antibodies and the antigen. This analysis identified Tyr-91 and Trp-96 in the light chain as hot spots for the interaction, and other residues made incremental contributions by conferring enthalpic advantages and reducing the dissociation rate constant. Systematic mutation of Tyr-91 indicated that CH-and -interactions between the aromatic ring at this site and the antigen made substantial contributions to the association, and van der Waals interactions inhibited dissociation, suggesting that aromaticity and bulkiness are critical for the specific recognition of polyether compounds by proteins.Ciguatera is a form of food poisoning caused by the ingestion of reef fish that have accumulated trace amounts of ciguatoxins of dinoflagellate origin via the food chain. More than 50,000 people suffer from ciguatera annually, making it one of the most common sources of food poisoning (1-4). The disease is characterized by gastrointestinal, neurological, and cardiovascular disturbances that often persist for months or years, and in severe cases, paralysis, coma, and death may occur (1). Ciguatoxins exert their effects by binding to voltage-sensitive sodium ion channels, causing persistent activation of the channels (1). Ciguatoxin and its congener, CTX3C, are structurally classified as ladder-like polyethers ( Fig. 1) (5, 6). A major obstacle to avoiding the disease is that ciguateric fish look, taste, and smell the same as uncontaminated fish. In addition, neither cooking nor freezing detoxifies the heat-stable ciguatoxins. Despite the seriousness of ciguatera, there is currently no rapid and reliable method for detecting these toxins at fisheries. The traditional method is a mouse bioassay of lipid extracts (7). Several additional methods for detecting ciguatoxins have recently been developed, including assays based on cytotoxicity (8), radioligand binding (8, 9), high performance chromatography (10), mass spectrometry (11-13), and an antibody-based immunoassay (14 -16). Among these, the antibody-based immunoassay is attractive, because it is accurate, sensitive, easily performed, and portable. Dur...

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Role of Tyr Residues in the Contact Region of Anti-lysozyme Monoclonal Antibody HyHEL10 for Antigen Binding

Cited by 68 publications

References 37 publications

How Oligomerization Contributes to the Thermostability of an Archaeon Protein

How Oligomerization Contributes to the Thermostability of an Archaeon Protein

Quantitative evaluation of the chicken lysozyme epitope in the HyHEL‐10 fab complex: Free energies and kinetics

Critical Contribution of Aromatic Rings to Specific Recognition of Polyether Rings

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