Structural Consequences of Mutations in Interfacial Tyr Residues of a Protein Antigen-Antibody Complex
2006; Elsevier BV; Volume: 282; Issue: 9 Linguagem: Inglês
10.1074/jbc.m605197200
ISSN1083-351X
AutoresMitsunori Shiroishi, Kouhei Tsumoto, Yoshikazu Tanaka, Akiko Yokota, Takeshi Nakanishi, Hidemasa Kondo, Izumi Kumagai,
Tópico(s)Enzyme Structure and Function
ResumoTyrosine is an important amino acid in protein-protein interaction hot spots. In particular, many Tyr residues are located in the antigen-binding sites of antibodies and endow high affinity and high specificity to these antibodies. To investigate the role of interfacial Tyr residues in protein-protein interactions, we performed crystallographic studies and thermodynamic analyses of the interaction between hen egg lysozyme (HEL) and the anti-HEL antibody HyHEL-10 Fv fragment. HyHEL-10 has six Tyr residues in its antigen-binding site, which were systematically mutated to Phe and Ala using site-directed mutagenesis. The crystal structures revealed several critical roles for these Tyr residues in the interaction between HEL and HyHEL-10 as follows: 1) the aromatic ring of Tyr-50 in the light chain (LTyr-50) was important for the correct ternary structure of variable regions of the immunoglobulin light chain and heavy chain and of HEL; 2) deletion of the hydroxyl group of Tyr-50 in the heavy chain (HTyr-50) resulted in structural changes in the antigen-antibody interface; and 3) the side chains of HTyr-33 and HTyr-53 may help induce fitting of the antibody to the antigen. Hot spot Tyr residues may contribute to the high affinity and high specificity of the antigen-antibody interaction through a diverse set of structural and thermodynamic interactions. Tyrosine is an important amino acid in protein-protein interaction hot spots. In particular, many Tyr residues are located in the antigen-binding sites of antibodies and endow high affinity and high specificity to these antibodies. To investigate the role of interfacial Tyr residues in protein-protein interactions, we performed crystallographic studies and thermodynamic analyses of the interaction between hen egg lysozyme (HEL) and the anti-HEL antibody HyHEL-10 Fv fragment. HyHEL-10 has six Tyr residues in its antigen-binding site, which were systematically mutated to Phe and Ala using site-directed mutagenesis. The crystal structures revealed several critical roles for these Tyr residues in the interaction between HEL and HyHEL-10 as follows: 1) the aromatic ring of Tyr-50 in the light chain (LTyr-50) was important for the correct ternary structure of variable regions of the immunoglobulin light chain and heavy chain and of HEL; 2) deletion of the hydroxyl group of Tyr-50 in the heavy chain (HTyr-50) resulted in structural changes in the antigen-antibody interface; and 3) the side chains of HTyr-33 and HTyr-53 may help induce fitting of the antibody to the antigen. Hot spot Tyr residues may contribute to the high affinity and high specificity of the antigen-antibody interaction through a diverse set of structural and thermodynamic interactions. Protein-ligand interactions via noncovalent bonds play a central role in many biological processes. To exhibit their functions, most proteins specifically recognize their partners or targets with certain affinities. Recently, analyses of the three-dimensional structures of many protein-protein complexes, mutational analyses, and analyses of the kinetics and thermodynamics of protein-protein interactions have revealed the roles of noncovalent bonds (i.e. hydrogen bonds, salt bridges, and van der Waals contacts). In general, the factors that determine the affinity and specificity of an interaction are the surface area and structure of the binding interface, including the electrostatic, hydrophilic, and hydrophobic complementarities (1Lo Conte L. Chothia C. Janin J. J. Mol. Biol. 1999; 285: 2177-2198Crossref PubMed Scopus (1785) Google Scholar). It has been reported that aromatic residues, especially Tyr and Trp, are more frequently found at the core of interaction sites and make more energetically favorable contributions to binding than do other amino acids (1Lo Conte L. Chothia C. Janin J. J. Mol. Biol. 1999; 285: 2177-2198Crossref PubMed Scopus (1785) Google Scholar, 2Bogan A.A. Thorn K.S. J. Mol. Biol. 1998; 280: 1-9Crossref PubMed Scopus (1656) Google Scholar, 3Chakrabarti P. Janin J. Proteins. 2002; 47: 334-343Crossref PubMed Scopus (503) Google Scholar). The concept of a protein-binding hot spot, defined as the critical residues for a protein interaction, was first proposed by Clackson and Wells (4Clackson T. Wells J.A. Science. 1995; 267: 383-386Crossref PubMed Scopus (1808) Google Scholar). Hot spot residues make energetically significant contributions to binding, typically contributing more than 2 kcal mol-1 in Gibbs energy change (ΔG) to the interaction. The frequency of Tyr residues in hot spots is far greater than that of Trp, suggesting that Tyr residues are a preferred amino acid for protein interaction hot spots (2Bogan A.A. Thorn K.S. J. Mol. Biol. 1998; 280: 1-9Crossref PubMed Scopus (1656) Google Scholar). Tyr has both a hydrophobic aromatic ring and a hydrophilic hydroxyl group in one side chain. Thus, from a structural point of view, Tyr residues can potentially contribute to binding via the following: 1) hydrogen bonding via the side-chain hydroxyl group; 2) hydrophobic interactions; 3) van der Waals interactions; and 4) amino-aromatic (cation-π) interactions via the aromatic ring (5Ma J.C. Dougherty D.A. Chem. Rev. 1997; 97: 1303-1324Crossref PubMed Scopus (3381) Google Scholar). Antibodies change the amino acid composition in their complementarity-determining regions (CDRs) 4The abbreviations used are: CDR, complementarity-determining region; HEL, hen egg white lysozyme; VH, variable region of immunoglobulin heavy chain; VL, variable region of immunoglobulin light chain; Fv, fragment of immunoglobulin variable regions; Fab, antigen-binding fragment of immunoglobulin; ITC, isothermal titration calorimetry; r.m.s.d., root-mean-square deviation. to bind specifically to various kinds of target antigens with high affinity. Tyr residues are commonly found in the CDRs of many antibodies (6Janin J. Chothia C. J. Biol. Chem. 1990; 265: 16027-16030Abstract Full Text PDF PubMed Google Scholar, 7Davies D.R. Cohen G.H. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 7-12Crossref PubMed Scopus (492) Google Scholar). Among the three-dimensional structures of 26 proteinaceous antigen-antibody and 14 peptide-antibody complexes, we observed that there were, on average, nearly 17 contacting residues in the antigen-binding site, of which 24% were Tyr. 5M. Shiroshi and K. Tsumoto, unpublished results. Thus, it appears that Tyr is used with the highest frequency during natural selection of antibodies. Tyr residues contribute to ∼17% of the overall interfacial area of antigen-antibody complexes (1Lo Conte L. Chothia C. Janin J. J. Mol. Biol. 1999; 285: 2177-2198Crossref PubMed Scopus (1785) Google Scholar), and buried Tyr residues tend to be interaction hot spots (2Bogan A.A. Thorn K.S. J. Mol. Biol. 1998; 280: 1-9Crossref PubMed Scopus (1656) Google Scholar). These observations suggest that Tyr residues are critical during the development of antibodies with high specificity and high affinity for their targets. Although Phe also has an aromatic ring, the frequency of Phe at antigen-binding sites was ∼4%, much lower than that of Tyr. 5M. Shiroshi and K. Tsumoto, unpublished results. In a recent report (8Binz H.K. Amstutz P. Kohl A. Stumpp M.T. Briand C. Forrer P. Grutter M.G. Pluckthun A. Nat. Biotechnol. 2004; 22: 575-582Crossref PubMed Scopus (548) Google Scholar), the ankyrin repeat protein off7, which was selected from a ribosome display library against maltosebinding protein, has a binding site, including seven aromatic residues, four of which are Tyr residues. In addition, the structure of artificially selected antibody against human vascular endothelial growth factor shows that antigen recognition is mediated mostly by Tyr residues, which cover 71% of the antigen-binding site (9Fellouse F.A. Wiesmann C. Sidhu S.S. Proc. Natl. Acad. Sci. U. S. A. 2004; 101: 12467-12472Crossref PubMed Scopus (251) Google Scholar). These results suggest that Tyr residues might play an important role in generating high affinity, high specificity binding sites during artificial selection processes as well. We have focused on the interaction between hen egg lysozyme (HEL) and the variable domain fragment (Fv) of the anti-HEL antibody HyHEL-10, which is one of best studied proteinaceous antigen-antibody interactions (10Tsumoto K. Ueda Y. Maenaka K. Watanabe K. Ogasahara K. Yutani K. Kumagai I. J. Biol. Chem. 1994; 269: 28777-28782Abstract Full Text PDF PubMed Google Scholar, 11Tsumoto K. Ogasahara K. Ueda Y. Watanabe K. Yutani K. Kumagai I. J. Biol. Chem. 1995; 270: 18551-18557Abstract Full Text Full Text PDF PubMed Scopus (69) Google Scholar, 12Tsumoto K. Ogasahara K. Ueda Y. Watanabe K. Yutani K. Kumagai I. J. Biol. Chem. 1996; 271: 32612-32616Abstract Full Text Full Text PDF PubMed Scopus (56) Google Scholar, 13Kondo H. Shiroishi M. Matsushima M. Tsumoto K. Kumagai I. J. Biol. 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Of the 19 residues in HyHEL-10 involved in antigen binding, six are Tyr residues, two in the variable regions of the immunoglobulin light chain (VL) and four in the heavy chain (VH) (Fig. 1, a and b) (13Kondo H. Shiroishi M. Matsushima M. Tsumoto K. Kumagai I. J. Biol. Chem. 1999; 274: 27623-27631Abstract Full Text Full Text PDF PubMed Scopus (65) Google Scholar, 19Padlan E.A. Silverton E.W. Sheriff S. Cohen G.H. Smith-Gill S.J. Davies D.R. Proc. Natl. Acad. Sci. U. S. A. 1989; 86: 5938-5942Crossref PubMed Scopus (496) Google Scholar). These Tyr residues contact 12 residues of HEL through van der Waals contacts, hydrogen bonding, or amino-aromatic (cation-π) interactions, or via interfacial water molecules. Together, these contacts encompass almost the entire HEL epitope recognized by HyHEL-10 (Fig. 1c). Because of the abundance of Tyr residues in the binding site, the HyHEL-10-HEL system may be a good model for investigating the role of Tyr residues in protein-protein interactions. In a previous study, we investigated the role of the Tyr residues in the VH region of HyHEL-10 (HyHEL-10 VH) using site-directed mutagenesis and isothermal titration calorimetry (ITC) (11Tsumoto K. Ogasahara K. Ueda Y. Watanabe K. Yutani K. Kumagai I. J. Biol. Chem. 1995; 270: 18551-18557Abstract Full Text Full Text PDF PubMed Scopus (69) Google Scholar). We found that Tyr residues located in the CDR of HyHEL-10 VH had a variety of significant roles in binding as follows: formation of hydrogen bonds via hydroxyl groups; hydrophobic interactions; van der Waals interactions (due to their large volume); and stabilization of local structure (due to their aromatic rings). Structural analyses of the complex between HEL and antibody mutants with one or two Tyr residues in the interfacial region of HyHEL-10 would provide more precise insights into the role of these residues in the antigen-antibody interaction. To elucidate the contribution of Tyr residues to high affinity and high specificity antigen binding, in combination with thermodynamic analyses we performed crystallographic studies of HyHEL-10 Fv mutants in complex with HEL, in which the six interfacial Tyr residues of HyHEL-10 were mutated to Phe and Ala. On the bases of the results obtained, we discuss how Tyr residues contribute to the specificity and affinity of proteinaceous antigen-antibody interactions. Preparation of HyHEL-10 Mutant Fvs—The gene structure of the expression vector of the HyHEL-10 Fv fragment was described in our previous paper (20Ueda Y. Tsumoto K. Watanabe K. Kumagai I. Gene (Amst.). 1993; 129: 129-134Crossref PubMed Scopus (40) Google Scholar). LY50A, 6The abbreviation used for mutants is, for example, LY50A, the mutant of HyHEL-10 Fv in which Ala is substituted for Tyr-50 of the VL chain. LY96F, and HY53A mutants were produced by the method of Kunkel as described in our previous paper (16Yokota A. Tsumoto K. Shiroishi M. Kondo H. Kumagai I. J. Biol. Chem. 2003; 278: 5410-5418Abstract Full Text Full Text PDF PubMed Scopus (60) Google Scholar, 21Kunkel T.A. Roberts J.D. Zakour R.A. Methods Enzymol. 1987; 154: 367-382Crossref PubMed Scopus (4868) Google Scholar). The double Ala substitution mutant of the HyHEL-10 Fv fragment, HY33A/Y53A, was generated by insertion of an XhoI/EcoRI fragment of the gene encoding the HY53A mutant into the identical site of the gene encoding the HY33A mutant. We obtained wild-type and mutant Fv fragments as secreted proteins using the Escherichia coli BL21(DE3) expression system. BL21(DE3) cells harboring the appropriate expression plasmid were precultured in 3 ml of LB medium, then inoculated into 2×YT medium with 100 mg/liter ampicillin, and shaken overnight at 28 °C. The culture was centrifuged, and the bacteria were resuspended in 2× YT medium containing 100 mg/liter ampicillin and isopropyl 1-thio-β-d-galactopyranoside at a final concentration of 1 mm and then shaken overnight at 28 °C again. The culture was centrifuged at 3000 × g for 20 min, and the supernatant was subjected to ammonium sulfate precipitation at 80% saturation, followed by centrifugation. The protein pellet was solubilized in 30-40 ml of PBS buffer and then dialyzed against PBS buffer. Fv fragments were purified using affinity chromatography. The protein solution was loaded onto an HEL-Sepharose column (20Ueda Y. Tsumoto K. Watanabe K. Kumagai I. Gene (Amst.). 1993; 129: 129-134Crossref PubMed Scopus (40) Google Scholar), and the column was washed with PBS buffer and then wash buffer (50 mm Tris-HCl (pH 8.5) containing 0.5 m NaCl). Fv fragments were eluted using elution buffer (0.1 m Gly-HCl (pH 2.0) containing 0.2 m NaCl) and buffered rapidly with 1 m Tris-HCl (pH 7.5). Fv-containing fractions were centrifuged, and minor impurities were removed by gel filtration with a Sephacryl S-200 column (GE Healthcare) pre-equilibrated with 50 mm Tris-HCl (pH 7.5) containing 0.2 m NaCl. Purified Fv fragments were concentrated using a Centriprep-10 column (Millipore, Bilerica, MA). The antigen, HEL, was purchased from Seikagaku-Kogyo Inc. (Tokyo, Japan). Isothermal Titration Calorimetry—Thermodynamic parameters of the interaction between HEL and wild-type or mutant HyHEL-10 Fv fragments were determined by ITC using a VPITC (MicroCal, Inc., Northampton, MA). The experimental conditions were as follows: in a calorimeter cell, the Fv fragment, at a concentration of 5 μm in 50 mm phosphate buffer (pH 7.2) containing 0.2 m NaCl, was titrated with a 50 μm solution of HEL in the same buffer at four different temperatures (25, 30, 35, or 40 °C). The HEL solution was injected 20 times in portions of 10 μl over 15 s. Thermograms were analyzed with the program Origin7 (MicroCal, Inc.) after subtraction of the thermogram against the buffer only. The enthalpy change (ΔH) and binding constant (Ka) for the antigen-antibody interaction were obtained directly from the experimental titration curve. The Gibbs free energy change (ΔG =-RT ln Ka) and the entropy change (ΔS = (-ΔG +ΔH)/T) for the association were calculated from ΔH and Ka. The heat capacity change (ΔCp) was estimated from the temperature dependence of the enthalpy change. Crystallization, Data Collection, and Structural Determination of the HyHEL-10 Mutant Fv-HEL Complex—Except for the HY33A/Y53A-HEL complex, all mutant Fv-HEL complexes were crystallized under conditions similar to those of the wild-type Fv-HEL complex (13Kondo H. Shiroishi M. Matsushima M. Tsumoto K. Kumagai I. J. Biol. Chem. 1999; 274: 27623-27631Abstract Full Text Full Text PDF PubMed Scopus (65) Google Scholar). The best crystals of Fv-HEL complexes were grown in 0.1 m Hepes buffer (pH 7.6-7.8), 9-11% w/v polyethylene glycol 6000, and 7-9% (w/v) 2-methyl-2,4-pentanediol. The resultant crystals were elongated bipyramid shapes. Crystals of the HY33A/Y53A-HEL complex were obtained in 0.1-0.2 m ammonium sulfate, 15-20% w/v polyethylene glycol 4000, and 0.1 m sodium acetate trihydrate (pH 4.6); hexagonal pillar-like crystals were obtained. All crystallization conditions included glycerol at a final concentration of 15% as a cryoprotectant. Data sets for the wild-type Fv-HEL complex and all mutant Fv-HEL complexes were obtained using the synchrotron x-ray source at beamline BL6A at the Photon Factory (Tsukuba, Japan). The diffraction images were processed by the interactive data processing package DPS/MOSFLM/CCP4. Integration was carried out using the MOSFLM software (22Leslie, A. G. W. (1992) Joint CCP4 + ESF-EAMCB Newsletter on Protein Crystallography, p. 26, Warrington, UKGoogle Scholar); scaling was carried out using SCALA software (23Evans P.R. Proceedings of CCP4 Study Weekend.1993: 114-122Google Scholar), and the final file of structural factors was obtained using TRUNCATE (24French S. Wilson K. Acta Crystallogr. Sect. A. 1978; 34: 517-525Crossref Scopus (908) Google Scholar) and MTZ2VARIOUS of the CCP4 suite (25Collaborative Computational ProjectActa Crystallogr. Sect. D Biol. Crystallogr. 1994; 50: 760-763Crossref PubMed Scopus (19878) Google Scholar). The structure of the Fv-HEL complexes was refined by using the CNS program (26Brunger A.T. Adams P.D. Clore G.M. DeLano W.L. Gros P. Grosse-Kunstleve R.W. Jiang J.S. Kuszewski J. Nilges M. Pannu N.S. Read R.J. Rice L.M. Simonson T. Warren G.L. Acta. Crystallogr. Sect. D. Biol. Crystallogr. 1998; 54: 905-921Crossref PubMed Scopus (17024) Google Scholar). The graphic program O (27Jones T.A. Zou J.Y. Cowan S.W. Kjeldgaard M. Acta Crystallogr. Sect. A. 1991; 47: 110-119Crossref PubMed Scopus (13036) Google Scholar) was used for making adjustments to the molecular model. Crystallographic and refinement data for each Fv mutant-HEL complex are summarized in the supplemental Table 1. Calculation of root-mean-square deviations (r.m.s.d.) for structural comparison was done using the programs LSQKAB (28Kabsch W. Acta Crystallogr. Sect. A. 1976; 32: 922-923Crossref Scopus (2456) Google Scholar) and COMPAR in the CCP4 software suite. Interfacial areas were calculated with NACCESS (29Hubbard S.J. Thornton J.M. NACCESS, Computer Program. University College London, London, UK1993Google Scholar). Shape complementarity scores were calculated with SC (30Lawrence M.C. Colman P.M. J. Mol. Biol. 1993; 234: 946-950Crossref PubMed Scopus (1122) Google Scholar) in the CCP4 suite. Determination of contacting atoms between Fv and HEL was performed with the CONTACT program in the CCP4 suite. Figures were drawn with the program WebLab Viewer Lite (Accelrys Inc., San Diego, CA). Atomic coordinates and structural factors for each mutant Fv-HEL complex have been deposited in the Protein Data Bank. The Protein Data Bank accession codes are as follows: wild type (2DQJ), LY50A (2DQI), HY33F (2DQC), HY33A/Y53A (2DQF), HY50F (2DQD), HY53A (2DQE), HY53F (2DQG), and HY58A (2DQH). Mutant Fv fragments were expressed using VL-VH coexpression vector and an E. coli expression system. They were purified to more than 95% purity by HEL-Sepharose affinity chromatography following size exclusion chromatography. As shown in our previous study, single Ala substitution mutants at positions H33 7The abbreviation used for residues is, for example, H33, residue number 33 of the VH chain. (HY33A mutant) and H50 (HY50A mutant) could not be purified by affinity chromatography, despite their high expression levels, because of their extremely low affinity for HEL (11Tsumoto K. Ogasahara K. Ueda Y. Watanabe K. Yutani K. Kumagai I. J. Biol. Chem. 1995; 270: 18551-18557Abstract Full Text Full Text PDF PubMed Scopus (69) Google Scholar). Ala substitutions at HTyr-33 and HTyr-50 cause large decreases in binding affinity using the HyHEL-10 Fab-HEL or HyHEL-63 Fab-HEL system; the latter antibody is related to HyHEL-10 and recognizes a similar epitope (17Pons J. Rajpal A. Kirsch J.F. Protein Sci. 1999; 8: 958-968Crossref PubMed Scopus (99) Google Scholar, 31Li Y. Urrutia M. Smith-Gill S.J. Mariuzza R.A. Biochemistry. 2003; 42: 11-22Crossref PubMed Scopus (57) Google Scholar). However, the double Ala substitution mutant of HTyr-33 and HTyr-53, HY33A/Y53A, could be purified using affinity chromatography. Thermodynamic Analyses—The interactions between mutant Fv fragments, constructed additionally in this study (LY50A, LY96F, HY33A/Y53A, and HY53A), and HEL were analyzed thermodynamically using ITC. The thermogram of each experiment was obtained by titrating the HEL solution into the Fv solution, from which the base line obtained by titrating HEL solution into the buffer was subtracted (Fig. 2, a-c). Together with the previous data on the other mutants, thermodynamic parameters are summarized in Table 1. In the Ala substitution mutant of LTyr-50 (LY50A), the thermodynamic analysis showed that the decrease in enthalpy change (ΔΔH, 10.4 kJ mol-1) was smaller than that of the Phe substitution mutant (ΔΔH, 16.7 kJ mol-1) (Fig. 2a and Table 1). In contrast, the entropic change was similar to that of the wild type (TΔΔS, 3.2 kJ mol-1). Ala substitution at HTyr-53 did not lead to the large Gibbs energy change compared with the wild-type-HEL interaction (ΔΔG; 0.9 kJ mol-1) (Fig. 2b and Table 1). However, large decreases in binding enthalpy (ΔΔH; 15.9 kJ mol-1) and entropic loss (TΔΔS; 15.0 kJ mol-1) were observed in the HY53A-HEL interaction. The binding constant of the HY33A/Y53A-HEL interaction (Ka = 1.9 × 106 m-1) was higher than that of the HY33A mutant but much lower than that of the wild type (Fig. 2c and Table 1). The large decrease in Gibbs energy (ΔΔG; 31.3 kJ mol-1) resulted from the large loss of binding enthalpy in HY33A/Y53A mutant.TABLE 1Thermodynamic parameters of mutant Fv-HEL interactions at 30 °C and pH 7.2MutantnKaΔGΔHΔΔHTΔSTΔΔSΔCp× 107 m−1kJ mol−1kJ mol−1 K−1Wild typeaData are from Ref. 11.0.9942−50.2−91.5−41.3−1.38LY50A0.992.6−43.0−81.110.4−38.13.2−1.98LY50FbData are from Ref. 16. ΔΔH and TΔΔS are the differences in binding enthalpy and entropy between mutant and wild type.1.0511−46.7−74.816.7−28.113.2−1.77LY96F1.012−46.8−94.6−3.1−47.8−6.5−1.97HY33AaData are from Ref. 11.,cHY33A and HY50A could not be purified by HEL-Sepharose affinity chromatography.HY33AY53A1.070.19−36.4−60.231.3−23.817.5NDHY33FaData are from Ref. 11.1.127.1−45.6−73.218.3−27.613.7−1.81HY50AaData are from Ref. 11.,cHY33A and HY50A could not be purified by HEL-Sepharose affinity chromatography.HY50FaData are from Ref. 11.1.052.9−43.1−59.831.7−16.724.6−1.38HY53A0.9630−49.3−75.615.9−26.315.0−1.58HY53FaData are from Ref. 11.1.0315−47.2−84.47.1−37.24.1−0.98HY58AaData are from Ref. 11.0.972.6−43.1−72.319.2−29.212.1−1.00HY58FaData are from Ref. 11.1.0123−48.5−85.36.2−36.84.5−1.58a Data are from Ref. 11Tsumoto K. Ogasahara K. Ueda Y. Watanabe K. Yutani K. Kumagai I. J. Biol. Chem. 1995; 270: 18551-18557Abstract Full Text Full Text PDF PubMed Scopus (69) Google Scholar.b Data are from Ref. 16Yokota A. Tsumoto K. Shiroishi M. Kondo H. Kumagai I. J. Biol. Chem. 2003; 278: 5410-5418Abstract Full Text Full Text PDF PubMed Scopus (60) Google Scholar. ΔΔH and TΔΔS are the differences in binding enthalpy and entropy between mutant and wild type.c HY33A and HY50A could not be purified by HEL-Sepharose affinity chromatography. Open table in a new tab Crystal Structure of Fv Mutant-HEL Complexes—The crystal structures of mutated HyHEL-10 Fv-HEL complexes were solved at resolutions sufficient for determining local structural differences (1.8-2.5 Ä) (supplemental Table 1). Most of the interfacial water molecules, which mediate the Fv-HEL interaction, were conserved between each mutant Fv-HEL complex and the wild-type complex (supplemental Table 2). Although LTyr-50 does not directly contact the residue of the VH chain, the relative orientation of VL, VH, and HEL is altered in the LY50A-HEL complex. In particular, the difference in the orientation of Fv and HEL between the wild-type and mutant complexes is large (Table 2). The large space generated by the Ala substitution at site L50 is occupied by water molecules that form a hydrogen-bonding network.TABLE 2r.m.s.d. in the C-α atoms of each chain (Ä)VL fitVH fitHEL fitFv fitAll fitLY50A vs. WTVL0.280.671.620.410.56VH0.810.200.980.310.37HEL1.681.040.331.110.6HY33F vs. WTVL0.070.090.160.080.09VH0.120.070.170.070.09HEL0.180.170.090.150.10HY33AY53A vs. WTVL0.301.151.980.440.76VH0.880.553.380.681.09HEL2.212.730.502.721.10HY50F vs. WTVL0.090.200.710.100.14VH0.180.120.820.130.17HEL0.460.450.120.420.24HY53F vs. WTVL0.130.140.220.130.14VH0.150.130.210.130.14HEL0.170.190.140.190.15HY53A vs. WTVL0.070.080.130.070.07VH0.090.070.160.070.08HEL0.090.090.070.090.08HY58A vs. WTVL0.140.200.290.150.15VH0.210.180.330.180.20HEL0.190.280.130.180.15 Open table in a new tab The structure of the HY33F-HEL complex shows that the overall structure, the interfacial water molecules, and the local structure around the mutation site of the HY33F-HEL complex are almost the same as those of the wild-type complex (Table 2 and supplemental Table 2 and Fig. 3b). The structure also showed that the decrease in polar area (-26 Ä2) and the increase in nonpolar area (+32 Ä2) that occurred at the site of H33 compared with the wild type were the result of the substitution of HTyr-33 with Phe (supplemental Table 3). In the HY50F-HEL complex structure, there are no overall structural differences compared with wild type (Table 2). Two hydrogen bonds are absent because of deletion of the hydroxyl group of HTyr-50, leading to large structural changes in the side chain of Arg-21 of HEL. The amino-aromatic (cation-π) interaction between HTyr-58 and Arg-21 of HEL is also absent, because of structural perturbation of the side chain of Arg-21 (Fig. 3c). In addition, there is a significant rearrangement of the interfacial water molecules. In the wild-type complex, a water molecule (Wat-10) is completely buried in the Fv-HEL interface and forms hydrogen bonds bridging the Fv-HEL interface. In the HY50F-HEL complex, although newly arranged water molecules (Wat-46, Wat-74, Wat-100, and Wat-250) form a network of hydrogen bonds at the Fv-HEL interface, some of these water molecules are exposed to solvent, suggesting that the network of hydrogen bonds contributes less energetically than that seen in the wild-type complex. Some hydrogen bonds, an amino-aromatic interaction, and many van der Waals interactions between Arg-21 of HEL and HTyr-58 are lost because of these structural changes (supplemental Table 4). In the HY58A-HEL complex structure, there are no large changes in overall structure or in the local structure of the mutation site (Table 2 and Fig. 3d). Two water molecules newly introduced at the mutated site make no hydrogen bonds bridging Fv and HEL. The substitution of Ala for HTyr-58 results in the loss of an amino-aromatic interaction, hydrogen bonding via a water molecule, and some van der Waals contacts. The structure of the HY53A-HEL complex shows that both the overall and local structures of the HY53A-HEL complex were similar to those of the wild-type-HEL complex (Table 2 and Fig. 3e). Notably, the B factor in the loop around Asp-101 of HEL in the HY53A-HEL complex is higher than that in the wild-type complex by 4.7 Ä2 compared with the overall B factor difference between the mutant and wild type (+0.7 Ä2). We solved the structure of the HY33A/Y53A-HEL complex from the trigonal crystals (space group P3221) obtained under conditions different from those for the wild-type and the other mutant complexes. Two complexes (HY33A/Y53A_1 and HY33A/Y53A_2) are present in an asymmetric unit (Fig. 4a). These two complexes have almost identical structures. The structure of each main chain of the mutant complex is similar to the corresponding main chain of the wild-type complex (average r.m.s.d. of each chain in the mutant complexes compared with that of the wild type was 0.30 Ä for VL, 0.55 Ä for VH, and 0.50 Ä for HEL; Table 2). However, large differences in the orientation of HEL to VL and/or VH were observed compared with the wild type. Furthermore, the r.m.s.d. of the main chain of HEL was 2.72 Ä when the Fv fragment of the mutant complex was superposed on that of the wild-type complex (Table 2 and Fig. 4, a and b). One salt bridge, some hydrogen bonds, and many van der Waals contacts were lost as a result of the removal of the side chains of HTyr-33 and HTyr-53 and the large change in the relative orientation between Fv and HEL (supplemental Table 4). Thus the large decrease in binding enthalpy in the HY33A/Y53A-HEL interaction may result from the loss of these bonds and contacts. Furthermore, these structural changes led to a decrease in the interfacial area and shape complementarity between Fv and HEL (supplemental Tables 3 and 5). When each chain of the mutant complex is compared with the corresponding chain of the wild-type complex, two large local structural differences are observed in the mutant complex. One is the large structural difference observed in the CDR-H2 loop by maximal 1.7 Ä (shift of the C-α atom at H53) (Fig. 4c). The other is the structure of the loop around Asp-101 of HEL. In the HY33A/Y53A complex, the loop stru
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