Critical Contribution of Aromatic Rings to Specific Recognition of Polyether Rings
2008; Elsevier BV; Volume: 283; Issue: 18 Linguagem: Inglês
10.1074/jbc.m710553200
ISSN1083-351X
AutoresKouhei Tsumoto, Akiko Yokota, Yoshikazu Tanaka, Mihoko Ui, Takeshi Tsumuraya, Ikuo Fujii, Izumi Kumagai, Yoko Nagumo, Hiroki Oguri, Masayuki Inoue, Masahiro Hirama,
Tópico(s)Neuroscience of respiration and sleep
ResumoTo 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 coli-expressed 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 1.7Å. 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-π and π-π 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. 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 coli-expressed 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 1.7Å. 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-π and π-π 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 (1Hirama M. Chem. Rec. 2005; 5: 240-250Crossref PubMed Scopus (51) Google Scholar, 2Scheuer P.J. Takahashi W. Tsutsumi J. Yoshida T. Science. 1967; 155: 1267-1268Crossref PubMed Scopus (240) Google Scholar, 3Scheuer P.J. Tetrahedron. 1994; 50: 3-18Crossref Scopus (185) Google Scholar, 4Lewis R.J. Toxicon. 2001; 39: 97-106Crossref PubMed Scopus (296) Google Scholar). 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 (1Hirama M. Chem. Rec. 2005; 5: 240-250Crossref PubMed Scopus (51) Google Scholar). Ciguatoxins exert their effects by binding to voltage-sensitive sodium ion channels, causing persistent activation of the channels (1Hirama M. Chem. Rec. 2005; 5: 240-250Crossref PubMed Scopus (51) Google Scholar). Ciguatoxin and its congener, CTX3C, are structurally classified as ladder-like polyethers (Fig. 1) (5Yasumoto T. Chem. Rec. 2001; 1: 228-242Crossref PubMed Scopus (342) Google Scholar, 6Murata M. Legrand A.M. Ishibashi Y. Yasumoto T. J. Am. Chem. Soc. 1989; 111: 8929-8931Crossref Scopus (308) Google Scholar). 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 (7Lewis R.J. Sellin M. Toxicon. 1993; 31: 1333-1336Crossref PubMed Scopus (47) Google Scholar). Several additional methods for detecting ciguatoxins have recently been developed, including assays based on cytotoxicity (8Manger R.L. Leja L.S. Lee S.Y. Hungerford J.M. Hokama Y. Dickey R.W. Granade H.R. Lewis R. Yasumoto T. Wekell M.M. J. AOAC Int. 1995; 78: 521-527Crossref PubMed Scopus (232) Google Scholar), radioligand binding (8Manger R.L. Leja L.S. Lee S.Y. Hungerford J.M. Hokama Y. Dickey R.W. Granade H.R. Lewis R. Yasumoto T. Wekell M.M. J. AOAC Int. 1995; 78: 521-527Crossref PubMed Scopus (232) Google Scholar, 9Dechraoui M.Y. Naar J. Pauillac S. Legrand A.M. Toxicon. 1999; 37: 125-143Crossref PubMed Scopus (224) Google Scholar), high performance chromatography (10Yasumoto T. Fukui M. Sasaki K. Sugiyama K. J. AOAC Int. 1995; 78: 574-582Crossref PubMed Scopus (97) Google Scholar), mass spectrometry (11Lewis R.J. Jones A. Toxicon. 1997; 35: 159-168Crossref PubMed Scopus (58) Google Scholar, 12Lewis R.J. Jones A. Vernoux J.P. Anal. Chem. 1999; 71: 247-250Crossref PubMed Scopus (95) Google Scholar, 13Yasumoto T. Igarashi T. Legrand A. Cruchet P. Chinain M. Fujita T. Naoki H. J. Am. Chem. Soc. 2000; 122: 4988-4989Crossref Scopus (166) Google Scholar), and an antibody-based immunoassay (14Oguri H. Hirama M. Tsumuraya T. Fujii I. Maruyama M. Uehara H. Nagumo Y. J. Am. Chem. Soc. 2003; 125: 7608-7612Crossref PubMed Scopus (80) Google Scholar, 15Tsumuraya T. Fujii I. Inoue M. Tatami A. Miyazaki K. Hirama M. Toxicon. 2006; 48: 287-294Crossref PubMed Scopus (48) Google Scholar, 16Nagumo Y. Oguri H. Tsumoto K. Shindo Y. Hirama M. Tsumuraya T. Fujii I. Tomioka Y. Mizugaki M. Kumagai I. J. Immunol. Methods. 2004; 289: 137-146Crossref PubMed Scopus (23) Google Scholar). Among these, the antibody-based immunoassay is attractive, because it is accurate, sensitive, easily performed, and portable. During the development of this immunoassay, extensive studies focused on preparing antibodies with high specificity for ciguatoxin, and recently a sandwich enzyme-linked immunosorbent assay using high affinity antibodies specific to both ends of CTX3C was established that can detect CTX3C down to the parts per billion level without cross-reactivity against other related marine toxins (14Oguri H. Hirama M. Tsumuraya T. Fujii I. Maruyama M. Uehara H. Nagumo Y. J. Am. Chem. Soc. 2003; 125: 7608-7612Crossref PubMed Scopus (80) Google Scholar, 15Tsumuraya T. Fujii I. Inoue M. Tatami A. Miyazaki K. Hirama M. Toxicon. 2006; 48: 287-294Crossref PubMed Scopus (48) Google Scholar), including brevetoxin A (17Shimizu Y. Chou H.N. Bando H. Duyne G.V. Clardy J. J. Am. Chem. Soc. 1986; 108: 514-515Crossref PubMed Scopus (363) Google Scholar), brevetoxin B (18Lin Y.Y. Risk M. Ray S.M. Van Engen D. Clardy J. Golik J. James J.C. Nakanishi K. J. Am. Chem. Soc. 1981; 103: 6773-6775Crossref Scopus (553) Google Scholar), okadaic acid (19Daranas A.H. Fernandez J.J. Norte M. Gavin J.A. Suarez-Gomez B. Souto M.L. Chem. Rec. 2004; 4: 1-9Crossref PubMed Scopus (19) Google Scholar), and maitotoxin (5Yasumoto T. Chem. Rec. 2001; 1: 228-242Crossref PubMed Scopus (342) Google Scholar). A monoclonal antibody, 1C49, was obtained from in vitro selection using a biotin-linked ABC ring fragment of CTX3C (CTX3C-ABC, 4The abbreviations used are: CTX3C-ABC, ABC ring compound of ciguatoxin CTX3C; CDR, complementarity-determining region; VH, variable region of immunoglobulin heavy chain; VL, variable region of immunoglobulin light chain; Fv, fragment of immunoglobulin variable regions (An example of the abbreviations used for mutants is L-Y91A, the mutant of the 1C49 Fv fragment in which Ala is substituted for Tyr-91 of the VL chain). Fig. 1) and an Fab library prepared from the spleens of mice immunized with CTX3C-ABC conjugated to keyhole limpet hemocyanin (16Nagumo Y. Oguri H. Tsumoto K. Shindo Y. Hirama M. Tsumuraya T. Fujii I. Tomioka Y. Mizugaki M. Kumagai I. J. Immunol. Methods. 2004; 289: 137-146Crossref PubMed Scopus (23) Google Scholar). Fab fragment of 1C49 binds CTX3C-ABC and CTX3C-ABCD with dissociation constants (Kd) of 8.6 × 10-8 m and 2.4 × 10-5 m, respectively (16Nagumo Y. Oguri H. Tsumoto K. Shindo Y. Hirama M. Tsumuraya T. Fujii I. Tomioka Y. Mizugaki M. Kumagai I. J. Immunol. Methods. 2004; 289: 137-146Crossref PubMed Scopus (23) Google Scholar). 1C49 also bound to CTX3C, although the affinity is reduced as compared with CTX3C-ABC (16Nagumo Y. Oguri H. Tsumoto K. Shindo Y. Hirama M. Tsumuraya T. Fujii I. Tomioka Y. Mizugaki M. Kumagai I. J. Immunol. Methods. 2004; 289: 137-146Crossref PubMed Scopus (23) Google Scholar). Elucidation of the molecular mechanism of the specific binding of the antibody to the ciguatoxin fragment would enable the antibody-based ciguatoxin immunoassay to be improved. Moreover, many marine toxins have polyether structures. These toxins include maitotoxin (5Yasumoto T. Chem. Rec. 2001; 1: 228-242Crossref PubMed Scopus (342) Google Scholar) and gambierol, which also cause ciguatera (5Yasumoto T. Chem. Rec. 2001; 1: 228-242Crossref PubMed Scopus (342) Google Scholar), brevetoxin, which causes neurotoxic shellfish poisoning (17Shimizu Y. Chou H.N. Bando H. Duyne G.V. Clardy J. J. Am. Chem. Soc. 1986; 108: 514-515Crossref PubMed Scopus (363) Google Scholar, 18Lin Y.Y. Risk M. Ray S.M. Van Engen D. Clardy J. Golik J. James J.C. Nakanishi K. J. Am. Chem. Soc. 1981; 103: 6773-6775Crossref Scopus (553) Google Scholar), dinophysistoxins, which cause diarrheic shellfish poisoning (19Daranas A.H. Fernandez J.J. Norte M. Gavin J.A. Suarez-Gomez B. Souto M.L. Chem. Rec. 2004; 4: 1-9Crossref PubMed Scopus (19) Google Scholar), palytoxin (20Shimizu Y. Nature. 1983; 302: 212Crossref PubMed Scopus (18) Google Scholar), and yessotoxin (21Takahashi T. Kusumi T. Kan Y. Satake M. Yasumoto T. Tetrahedron Lett. 1996; 37: 7087-7090Crossref Scopus (86) Google Scholar). Therefore, research focused on antibodies that can recognize polyether compounds may produce a more effective antibody-based immunoassay based on well understood mechanisms. Here, we investigated the binding of 1C49 to the CTX3C-ABC polyether from a structural viewpoint. First, we resolved the crystal structure of the 1C49 Fv fragment in complex with CTX3C-ABC at a resolution of 1.7 Å. CTX3C-ABC is deeply buried in the binding pocket and interacts with 1C49 by using three hydrogen bonds and many van der Waals interactions. Kinetic analyses of mutant proteins indicated that L-Tyr-91 and L-Trp-96 are critical residues, i.e. hot spots, for the interaction. Systematic mutation of L-Tyr-91 showed that aromatic interactions and the bulkiness by L-Tyr-91 are essential for the association and the inhibition of dissociation, respectively. Construction of an Expression Vector for the Fv Fragment of Monoclonal Antibody 1C49 and Its Mutant Proteins—The gene encoding a fragment of the immunoglobulin variable regions (Fv) was amplified with KOD-Plus DNA polymerase (Toyobo) by using a vector expressing the Fab fragment of 1C49 (16Nagumo Y. Oguri H. Tsumoto K. Shindo Y. Hirama M. Tsumuraya T. Fujii I. Tomioka Y. Mizugaki M. Kumagai I. J. Immunol. Methods. 2004; 289: 137-146Crossref PubMed Scopus (23) Google Scholar) as a template. To amplify a fragment of the variable region of the immunoglobulin heavy chain (VH), we used the following primer sets (restriction enzyme sites are underlined): 1C49-VH-NcoI-back (5′-NNNCCATGGCCCAGGTGCAGCTGCTCGAGTCTGGGGCTGA-3′), 1C49-VH-SacII-forward (5′-NNNCCGCGGAAGAGACGGTGACTGAGG-3′). The PCR products were digested by NcoI and SacII and then inserted into a T7 promoter-based expression vector (22Makabe K. Asano R. Ito T. Tsumoto K. Kudo T. Kumagai I. Biochem. Biophys. Res. Commun. 2005; 328: 98-105Crossref PubMed Scopus (22) Google Scholar) to attach a His tag at the C terminus and a pel-B signal sequence at the N terminus of VH. A fragment of the variable region of the light chain (VL) was first amplified with the primer set 1C49-VL-NcoI-back (5′-NNNCCATGGCCGACATTCAGATGACACAGTCTC-3′), 1C49-VL-SacII-forward (5′-NNNCCGCGGCCCGTTTGATTTCCAGATTG-3′) (restriction enzyme sites are underlined), and the product was also inserted into the T7 promoter-based expression vector to attach a His tag at the C terminus and a pel-B signal sequence at the N terminus. Subsequently, the His-tagged VL fragment was amplified by using the vector resulting from the first amplification and the primer set SpeI-pel-B-back (5′-CACTAGTTATTTCAAGGAGACAGTCATAATGAAATACC-3′) and BamHI-His tag-forward (5′-NNNGGATCCGCTATTAATGGTGGTGATGATGGTG-3′), followed by digestion by SpeI and BamHI. By inserting the product downstream of the VH-expressing vector described above, we created an expression vector with a gene for the Fv fragment where the genes for VH and VL are connected in tandem. pel-B signal sequences and His tags were attached to the N and C termini of both chains, respectively. The expression vector is shown in supplemental Fig. S1. The expression vectors for the mutant proteins were constructed by amplifying a DNA fragment by the two-stage PCR method using synthesized primers designed for each mutation, and inserting these instead of the VH or VL fragment described above into the vectors. The accuracy of the DNA sequences was confirmed by sequencing using an ABI 310 Genetic Analyzer (Applied Biosystems, Tokyo, Japan). Expression and Purification of the 1C49 Fv Fragment and Its Mutant Proteins—Transformed Escherichia coli strain BL21(DE3) harboring the expression vector for the desired protein was grown until the early stationary phase at 28 °C in LB medium supplemented with 100 μg ml-1 ampicillin. To induce expression of the desired protein, isopropyl-β-d-thiogalactopyranoside was added to a final concentration of 1 mm, and the culture was continued for 14 h. The culture was centrifuged at 5000 × g for 15 min at 4 °C, and the supernatant was subjected to ammonium sulfate precipitation at 60% saturation, followed by centrifugation at 7000 × g for 30 min at 4 °C. The protein pellet was solubilized in 30–40 ml of 20 mm Tris-HCl (pH 8.0), 500 mm NaCl, and then dialyzed against the same buffer. Fv fragments were purified with His Bind Resin (Novagen) previously charged with NiSO4. The protein solution was loaded onto the column, and the column was washed with wash buffer (20 mm Tris-HCl (pH 8.0), 500 mm NaCl, 20 mm imidazole). Fv fragments were eluted with elution buffer (20 mm Tris-HCl (pH 8.0), 500 mm NaCl, 300 mm imidazole). Fractions containing Fv fragments were dialyzed against 50 mm phosphate buffer (pH 6.5) and then further purified on a Resource S column (GE Healthcare Biosciences AB) equilibrated with the same buffer. Absorbed proteins were eluted by using a 0–0.8 m gradient of NaCl in 50 mm phosphate (pH 6.5). Fractions containing the Fv fragments were collected and used for further experiments. For the H-W33A, L-Y91L, and L-Y91V mutants, because of the insolubility of the expressed proteins they were prepared by refolding from the insoluble fraction of cells with a stepwise dialysis system described previously (23Tsumoto K. Shinoki K. Kondo H. Uchikawa M. Juji T. Kumagai I. J. Immunol. Methods. 1998; 219: 119-129Crossref PubMed Scopus (186) Google Scholar, 24Umetsu M. Tsumoto K. Hara M. Ashish K. Goda S. Adschiri T. Kumagai I. J. Biol. Chem. 2003; 278: 8979-8987Abstract Full Text Full Text PDF PubMed Scopus (155) Google Scholar, 25Tsumoto K. Ejima D. Kumagai I. Arakawa T. Protein Expr. Purif. 2003; 28: 1-8Crossref PubMed Scopus (356) Google Scholar). Crystallization of the 1C49 Fv Fragment—Purified 1C49 Fv fragment was dialyzed against 10 mm Tris-HCl (pH 8.0) and then concentrated to 10.5 mg ml-1. CTX3C-ABC solution (10 mm in DMSO) was added to the protein solution to a final molar ratio of protein:CTX3C-ABC of 1:1.2, followed by a static incubation at room temperature for 1 h. The initial crystallization conditions were screened by the sparse matrix method at 20 °C, using a Crystal Screen kit and Crystal Screen2 kit (Hampton Research) and Wizard I and Wizard II (Emerald Biostructures, Bainbridge Island, WA). Crystals of the 1C49 Fv fragment in complex with CTX3C-ABC most suitable for further analyses were grown from 0.1 m MES buffer (pH 5.7) containing 25% polyethylene glycol monomethyl ether 5000 and 2 m ammonium sulfate by the hanging drop, vapor-diffusion method. Diffraction Data Collection and Processing—X-ray diffraction data were collected on the beamline NW12 at Photon Factory (Tsukuba, Japan) under cryogenic conditions (100 K). Crystals were soaked in a crystallization buffer containing 15% glycerol before mounting. Diffraction data were collected up to a resolution of 1.7 Å. The data were indexed and integrated with the program HKL2000 (26Otwinowski Z. Minor W. Methods Enzymol. 1997; 276: 307-326Crossref PubMed Scopus (38594) Google Scholar) and were scaled and merged with the program SCALEA (27Weiss M. J. Appl. Crystallogr. 2001; 34: 130-135Crossref Scopus (584) Google Scholar) within the CCP4 program suite (28Acta Crystallogr D Biol Crystallogr. 1994; 50: 760-763Crossref PubMed Scopus (19793) Google Scholar). The data collection and processing statistics are summarized in Table 1.TABLE 1Data collection and refinement statisticsData collectionSpace groupI212121Cell dimensions (Å)a = 47.2, b = 97.5, c = 115.7BeamlinePF NW12Resolution (Å)aThe values in parentheses refer to data in the highest resolution shell50-1.70 (1.79-1.70)Wavelength (Å)1.0000Rsym (%)aThe values in parentheses refer to data in the highest resolution shell,bRsym = ΣhΣi|Ih,i – 〈Ih〉|/ΣhΣi|Ih,i|, where 〈Ih〉 is the mean intensity of a set of equivalent reflections5.8 (15.2)Completeness (%)aThe values in parentheses refer to data in the highest resolution shell98.8 (95.8)Observed reflections203,645Unique reflections29,463I/σ (I)25.7 (9.6)MultiplicityaThe values in parentheses refer to data in the highest resolution shell6.9 (6.5)Refinement and model qualityResolution range (Å)aThe values in parentheses refer to data in the highest resolution shell20–1.70 (1.81–1.70)No. of reflections29,447R-factoraThe values in parentheses refer to data in the highest resolution shell,cR-factor = Σ|Fobs – Fcalc|/ΣFobs, where Fobs and Fcalc are the observed and calculated structure factor amplitudes0.198 (0.207)Rfree-factoraThe values in parentheses refer to data in the highest resolution shell,dRfree-factor was calculated for the R-factor for a random 5% subset of all reflections0.210 (0.249)Total protein atoms1,767Total ligand atoms32Total water atoms196Average B-factor (Å2)20.5Root mean square deviation from idealBond lengths (Å)0.005Bond angles (°)1.4Ramachandran plotResidues in most favored regions (%)87.2Residues in additional allowed regions (%)12.2Residues in generously allowed regions (%)0.0Residues in disallowed regions (%)0.5a The values in parentheses refer to data in the highest resolution shellb Rsym = ΣhΣi|Ih,i – 〈Ih〉|/ΣhΣi|Ih,i|, where 〈Ih〉 is the mean intensity of a set of equivalent reflectionsc R-factor = Σ|Fobs – Fcalc|/ΣFobs, where Fobs and Fcalc are the observed and calculated structure factor amplitudesd Rfree-factor was calculated for the R-factor for a random 5% subset of all reflections Open table in a new tab Structure Solution and Refinement—The structure of the 1C49 Fv fragment in complex with CTX3C-ABC was determined by the molecular replacement method using the program CNS (29Brunger 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 (16978) Google Scholar). VH and VL regions of an Fab (PDB codes 1AXS and 1FJ1) were used as search models for VH and VL, respectively. An electron density derived from CTX3C-ABC was observed at the CDR region (supplemental Fig. S2). The structure coordinates of CTX3C-ABC were prepared with the program Chem Office 6.0 (CambridgeSoft, Cambridge, MA), and the topology file and the parameter file of CTX3C-ABC were prepared at the HIC-UP web site (30Kleywegt G.J. Jones T.A. Acta Crystallogr. Sect. D Biol. Crystallogr. 1998; 54: 1119-1131Crossref PubMed Scopus (496) Google Scholar). To monitor the refinement, a random 5% subset of all reflections was set aside for calculation of the Rfree factor. The positional and individual B factor refinements were carried out automatically with the program LAFIRE (31Yao M. Zhou Y. Tanaka I. Acta Crystallogr. Sect. D Biol. Crystallogr. 2006; 62: 189-196Crossref PubMed Scopus (74) Google Scholar). After automatic refinement and model fitting by LAFIRE, several cycles of refinement with the program CNS and manual model fitting were carried out. Finally, the water molecules were picked automatically, and then ligand molecules were placed manually. The crystallographic R values and Rfree values converged to 19.8% and 21.0%, respectively. The stereochemical quality of the final refined models was analyzed by the program PROCHECK (32Laskowski R.A. MacArthur M.W. Moss D.S. Thornton J.M. J. Appl. Crystallogr. 1993; 26: 283-291Crossref Google Scholar). The refinement statistics are summarized in Table 1. Residues are numbered, and framework regions and complementarity-determining regions (CDRs) are designated according to Kabat et al. (33Kabat E.A.Wu T.T., Perry, H. Gottesman, K., and Foeller, C. (1991) Sequences of Proteins of Immunological Interest, Fifth Ed., National Institutes of Health, Bethesda, MDGoogle Scholar). The atoms that form the contact between 1C49 Fv and CTX3C-ABC were identified with the CONTACT program in the CCP4 suite, in which the length thresholds of C-C, C-N, C-O, O-O, O-N, and N-N are 4.1, 3.8, 3.7, 3.3, 3.4, and 3.4 Å, respectively. The figures were drawn with the program PyMOL (DeLano Scientific LLC). The surface area was calculated with the program AREAIMOL in the CCP4 suite using a probe radius of 1.4 Å. Analyses of the Interaction between CTX3C-ABC and the 1C49 Fv Fragment or Its Mutants—The interaction between CTX3C-ABC and the 1C49 Fv fragment or its mutant proteins were analyzed by surface plasmon resonance (SPR) spectroscopy with BIACORE 2000 (GE Healthcare Bio-Science AB, Uppsala, Sweden). Bovine serum albumin-conjugated CTX3C-ABC was immobilized onto the cells in a CM5 sensor chip. Various concentrations of the 1C49 Fv fragment or its mutants in HBS-EP buffer (10 mm HEPES (pH 7.4), 150 mm NaCl, 3.4 mm EDTA, 0.05% surfactant P20) were flowed over the CTX3C-ABC. The data were normalized by subtracting the response from a blank cell in which bovine serum albumin alone was immobilized. BIAevaluation software (GE Healthcare Bio-Science AB) was used to analyze the data. Kinetic parameters were calculated by a global fitting analysis with the assumptions of the 1:1 Langmuir binding model. The association constants of L-Y91A and L-Y91S mutants were calculated by using a Scatchard plot because of their fast dissociation rate constants. Crystal Structure of the 1C49 Fv Fragment in Complex with CTX3C-ABC—We solved the crystal structure of the 1C49 Fv fragment in complex with CTX3C-ABC by the molecular replacement method at a resolution of 1.7 Å. The CDRs of the antibody formed a cavity around the ligand binding site, and the antigen, CTX3C-ABC, lay in the cavity (Fig. 2, A and B). A number of aromatic residues, H-Tyr-100a, L-Tyr-49, L-Tyr-91, H-Trp-33, L-Trp-96, and L-Phe-94, were located around the ligand binding site and contributed to ligand binding by van der Waals interactions (Fig. 2C). However, there were no polar residues around the binding site, suggesting that hydrogen bonds or ionic interactions have little effect. Fig. 2B represents an electrostatic surface of 1C49 viewed from above the ligand binding site. Although the entrance of the cavity is charged, the inside is relatively noncharged, also suggesting that van der Waals interactions have greater effects than electrostatic interactions. Ether groups in CTX3C-ABC do not act as proton acceptors for hydrogen bonding despite their abundance. CTX3C-ABC is deeply buried in the cavity, with 457 Å2 of its 529 Å2 surface area (86.4%) within the cavity, suggesting a well organized shape complementarity. The contacts between 1C49 and CTX3C-ABC observed in the crystal structure are listed in supplemental Table S1. CTX3C-ABC has 62 interactions with 15 residues, which are mainly in CDRs. Despite these extensive interactions, only three residues, H-Gln-35, H-Tyr-100a, and L-Trp-96, formed hydrogen bonds, again suggesting the predominance of van der Waals interactions. The binding of CTX3C-ABC is achieved mainly through many aromatic rings, such as those in Trp and Tyr. The three water molecules (Wat) that construct hydrogen bonds with both CTX3C-ABC and 1C49 are observed at the interface (Table 2). Wat-8 and Wat-10 are exposed to solvent above the ligand binding pocket, whereas Wat-50 is located inside the cavity and fills the interface between CTX3C-ABC and the antibody. Fig. 3 is a schematic of the residues around the ligand.TABLE 2Contacts between 1C49 and CTX3C-ABC via water moleculesWater moleculeInteracting atomDistanceResidueAtomÅWat-8CTX3C-ABCO242.66H-W33O2.86Wat-10CTX3C-ABCO232.86L-Lys-32Nζ2.98L-Tyr-91O2.75L-Phe-94Nζ2.96Wat-50CTX3C-ABCO113.64H-Gln-35N∈22.83H-Ser-99O2.79 Open table in a new tab Kinetic Analysis of the Interaction between CTX3C-ABC and the 1C49 Fv Fragment—We used SPR to explore the kinetics of the interaction between CTX3C-ABC and the 1C49 Fv fragment. 1C49 Fv was injected into flow cells on which bovine serum albumin-conjugated CTX3C-ABC was immobilized. We applied a global curve-fitting analysis to the SPR sensorgrams to generate response curves (Fig. 4A) using the assumptions of the Langmuir binding model and a stoichiometry of 1:1. We then used these responses to determine the association rate constant and dissociation rate constant (kon and koff) of the interaction between CTX3C-ABC and the 1C49 Fv fragment at 10 °C; kon was determined to be 7.77 × 105 m-1s-1, and koff was determined to be 3.50 × 10-4 s-1, resulting in an association constant (KA) of 2.22 × 109 m-1 (Table 3). The fast association rate constant, slow dissociation rate constant, and resulting high association constant are typical of antigen-antibody interactions. To obtain the activation energy for the interaction of CTX3C-ABC with the 1C49-Fv fragment, the kinetic analyses were performed at a range of temperatures (10, 15, 20, and 25 °C). On the basis of the temperature dependence of the association constant (Fig. 4B), the van't Hoff enthalpy (ΔHvan't Hoff) was calculated to be -31.2 kJ m-1, suggesting that this interaction is enthalpy-driven.TABLE 3Kinetic parameters at 10 °C and van't Hoff enthalpy of wild-type and mutant 1C49 Fv fragmentskonaThese values were obtained with a global curve fitting analysis of the SPR sensorgrams with the assumptions of the Langmuir binding model and a stoichiometry of 1:1koffaThese values were obtained with a global curve fitting analysis of the SPR sensorgrams with the assumptions of the Langmuir binding model and a stoichiometry of 1:1KAΔHvan't Hoffbvan't Hoff enthalpies were obtained from the slope of the van't Hoff plot by the equation ln KD = ΔH/RT – ΔS/Rm–1 s–1s–1m–1kJmol–1WT7.77 × 1053.50 × 10–42.22 × 109–31.2L-Y49A1.03 × 1068.06 × 10–41.28 × 109–28.4L-Y91AcThe association constant was determined by a steady-state fit of the binding data from maximal equilibrium values of the SPR sensorgrams3.55 × 106–9.23L-F94A5.53 × 1052.30 × 10–32.41 × 108–16.0L-W96ANDdND, no detectable bindingNDNDNDH-S99A5.19 × 1054.88 × 10–31.06 × 108–24.7H-D100A1.48 × 1055.70 × 10–32.60 × 107–28.2H-W33A7.93 × 1051.35 × 10–25.87 × 107–8.60H-Q35A1.54 × 1055.58 × 10–32.67 × 107–20.7H-Y100aA8.21 × 1053.74 × 10–42.20 × 109–26.9a These values were obtained with a global curve fitting analysis of the SPR sensorgrams with the assumptions of the Langmuir binding model and a stoichiometry of 1:1b van't Hoff enthalpies were obtained from the slope of the van't Hoff plot by the equation ln KD = ΔH/RT – ΔS/Rc The association constant was determined by a steady-state fit of the binding data from maximal equilibrium values of the SPR sensorgramsd ND, no detectable binding Open table in a new tab Effects of Mutating Residues around the CTX3C-ABC Binding Site—To investigate the contribution of residues around the CTX3C-ABC binding site, we created Ala mutants of L-Y49, L-Y91, L-F94, L-W96, H-S99, H-D100, H-W33, H-Q35, and H-Y100a, which are residues in CDR regions and interact with plural atoms in CTX3C-ABC, and used SPR to evaluate their binding affinities to CTX3C-ABC (supplemental Fig. S3). Because of the fast dissociation rate constant, the association constant of the L-Y91A mutant was determined by a Sc
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