Critical Contribution of Aromatic Rings to Specific Recognition of Polyether Rings: THE CASE OF CIGUATOXIN CTX3C-ABC AND ITS SPECIFIC ANTIBODY 1C49

Kouhei Tsumoto,Akiko Yokota,Yoshikazu Tanaka,Mihoko Ui,Takeshi Tsumuraya,Ikuo Fujii,Izumi Kumagai,Yoko Nagumo,Hiroki Oguri,Masayuki Inoue,Masahiro Hirama

doi:10.1074/jbc.m710553200

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.

Highlights

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
During the development of this immunoassay, extensive studies focused on preparing antibodies with high specificity for ciguatoxin, and recently a sandwich enzymelinked 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 [14, 15], including brevetoxin A [17], brevetoxin B [18], okadaic acid [19], and maitotoxin [5]
Fab fragment of 1C49 binds CTX3C-ABC and underlined), and the product was inserted into the T7 proCTX3C-ABCD with dissociation constants (Kd) of 8.6 ϫ 10Ϫ8 M moter-based expression vector to attach a His tag at the C terand 2.4 ϫ 10Ϫ5 M, respectively [16]. 1C49 bound to CTX3C, minus and a pel-B signal sequence at the N terminus

Summary

H O H Me

LO M (Fv) was amplified with KOD-Plus DNA polymerase (Toyobo) by using a vector expressing the Fab fragment of 1C49 [16] as a template. Fab fragment of 1C49 binds CTX3C-ABC and underlined), and the product was inserted into the T7 proCTX3C-ABCD with dissociation constants (Kd) of 8.6 ϫ 10Ϫ8 M moter-based expression vector to attach a His tag at the C terand 2.4 ϫ 10Ϫ5 M, respectively [16]. Elucidation of the molecular mechanism of the specific bind- vector resulting from the first amplification and the primer set ing of the antibody to the ciguatoxin fragment would enable the SpeI-pel-B-back The expression ing of 1C49 to the CTX3C-ABC polyether from a structural view- vectors for the mutant proteins were constructed by amplifying point. CTX3C-ABC is deeply buried in the binding pocket and interacts instead of the VH or VL fragment described above into the with 1C49 by using three hydrogen bonds and many van der Waals vectors.

EXPERIMENTAL PROCEDURES

RESULTS

DISCUSSION