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SELECTIVITY AND BINDING OF CATALYTIC ANTIBODIES
Fabio Benedetti, Dipartimento di Scienze Chimiche, Università di Trieste.
Homology modelling is a useful tool for the study of antibody structures, when crystallographic studies are not available. Thanks to the high homology between sequences - and structures - of antibodies from the same species, homology models reproduce in general with reasonable fidelity the actual crystallographic structure of the protein and can thus be used to describe the relationships between structure and function of antibodies.
The application of homology modelling to the study of the catalytic mechanism of two hydrolytic antibodies will be here described.
ScFv antibody 522c2 was obtained from a catalytic antibody raised in BalbC mice against a phosphonate hapten mimicking the transition state for hydrolysis of tyrosine benzoate. The original 522c2 antibody accelerates the hydrolysis of tyrosine benzoate with kcat/kuncat = 15000 and displays the same activity towards simple tyrosine containing peptides.
Preliminary experiments indicate that this catalytic activity is retained by the ScFv antibody. A homology model was obtained from the sequence of the ScFv, using antibody 1DFV as a model for the heavy chain, while the light chain was built from homologous antibodies 1NBV (fragments 1-90) and 1IGT (fragments 91-117). The structure thus obtained was then docked with the phosphonate transition state analog, revealing an oxyanion hole-shaped binding site (Fig. 1), with hydrophobic residues surrounding the phenyl ring, a tyrosine and an arginine within H-bond distance of the phosphonate oxygens and two more arginines in proximity of the negatively charged hapten.
This mode of binding supports the hypothesis that transition state stabilization by the oxyanion hole is an important component of the catalytic activity of 522c2 ScFv.
Figure 1. Homology model of 522c2 ScFv
Murine IgG 312D6 was obtained against a KLH conjugate of N-tolylsulfonyl indole and catalyzes the hydrolysis of N-benzoyl indoles, including N'-benzoyl tryptophan, with rate acceleration factors kcat/kuncat of 400-800. Despite the modest catalytic activity, the antibody is interesting because it is one of the very few abzymes with amidase activity reported so far. A fully functional Fab fragment was obtained by papain hydrolysis of the antibody; however, attempts to crystallize the Fab in the presence of the sulfonamide hapten failed and this prompted us to resort to modelling once more. To this end the sequences of light and heavy chains corresponding to the binding site (approximately the final 100 residues of each N-terminal) were determined from the corresponding gene sequences and a homology model was built (Fig. 2a). Docking of the sulfonamide antigen on the protein, however, gave unrealistic results and thus the model structure was analyzed with GRID,1 in order to locate possible site(s) of binding, using the DRY and SULFONAMIDE probes. In Fig. 2a areas of interaction with the DRY probe are reported as pink surfaces and extend over the whole interface between heavy and light chain, while cyan surfaces represent areas of interaction with the SULFONAMIDE probe which clearly identify the antigen-binding site. An expansion of this region is reported in Fig. 2b which shows that the binding site consists of a hydrophobic hole lined by Trp (blue) and Phe residues (cyan) with two Arg residues in proximity of the opening, presumably involved in electrostatic interactions with the sulfonamide oxygens. This suggests that the catalytic activity of antibody 312D6 could again be due to stabilization of the negatively charge tetrahedral intermediate by an oxyanion hole.
Figure 2. Antibody 312D6. a) GRID analysis of the homology model. b) Binding site.
1. Goodford, P.J. J. Med. Chem. 1985, 28, 849. Bobbyer, D.N.A. et al. J. Med. Chem. 1989, 32, 1083
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