title: Critical contribution of aromatic rings to specific recognition of polyether rings. The case of ciguatoxin CTX3C-ABC and its specific antibody 1C49.
abstract: 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 1.7A. 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-pi and pi-pi 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.
authors.affiliation: Department of Medical Genome Sciences, Graduate School of Frontier Sciences, The University of Tokyo, Kashiwa 277-8562, Japan. tsumoto@k.u-tokyo.ac.jp
authors.forename: Kouhei
authors.initials: K
authors.lastname: Tsumoto
authors.forename: Akiko
authors.initials: A
authors.lastname: Yokota
authors.forename: Yoshikazu
authors.initials: Y
authors.lastname: Tanaka
authors.forename: Mihoko
authors.initials: M
authors.lastname: Ui
authors.forename: Takeshi
authors.initials: T
authors.lastname: Tsumuraya
authors.forename: Ikuo
authors.initials: I
authors.lastname: Fujii
authors.forename: Izumi
authors.initials: I
authors.lastname: Kumagai
authors.forename: Yoko
authors.initials: Y
authors.lastname: Nagumo
authors.forename: Hiroki
authors.initials: H
authors.lastname: Oguri
authors.forename: Masayuki
authors.initials: M
authors.lastname: Inoue
authors.forename: Masahiro
authors.initials: M
authors.lastname: Hirama
mesh_terms: D019943:Amino Acid Substitution; D000596:Amino Acids; D000906:Antibodies / Q000737:chemistry / Q000276:immunology*; D000911:Antibodies, Monoclonal / Q000737:chemistry / Q000276:immunology*; D000918:Antibody Specificity / Q000276:immunology*; D000936:Antigen-Antibody Complex / Q000737:chemistry; D001666:Binding Sites, Antibody; D002922:Ciguatoxins / Q000737:chemistry / Q000276:immunology*; D018360:Crystallography, X-Ray; D006841:Hydrocarbons, Aromatic / Q000737:chemistry*; D007128:Immunoglobulin Fragments / Q000737:chemistry; D007135:Immunoglobulin Variable Region / Q000737:chemistry; D007700:Kinetics; D008024:Ligands; D008958:Models, Molecular; D009154:Mutation / Q000235:genetics; D013816:Thermodynamics
chemical_list: D000596:Amino Acids; D000906:Antibodies; D000911:Antibodies, Monoclonal; D000936:Antigen-Antibody Complex; D006841:Hydrocarbons, Aromatic; D007128:Immunoglobulin Fragments; D007135:Immunoglobulin Variable Region; D008024:Ligands; C466858:ciguatoxin 3C; D002922:Ciguatoxins