The molecular recognition of proteins by antibodies: A model for rational design

Basic Sciences

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Abstract

The Structural Immunology Section investigates molecular recognition in antibody complexes with proteins as model systems to elucidate the general principles of protein target recognition by antibodies. The results to date have provided new paradigms on the mechanism of antibody-antigen binding, and in addition have provided new methodology for examining molecular interaction networks. We have developed novel protocols for surface plasmon resonance (SPR) analysis which reveal that (i) Antibody-antigen bimolecular association is a time-dependent 2-step binding process; (ii) Kinetics, thermodynamics, and water movements accompanying the 2 steps are distinctly different, and define which of the steps are rate limiting, information which informs the effective design of competitive inhibitors; (iii) Antibody affinity maturation, a protype of molecular evolution, is driven by thermodynamics, thus thermodynamics inform the rational design of antibodies; (iv) Affinity and specificity of protein-protein interactions are determined by inherent protein flexibility, thus receptor and ligand flexibility must be considered in structure-based drug discovery; (v) Intramolecular salt link networks provide strong electrostatic interactions which can be significantly stabilizing and can modulate the dynamics of antibody recognition and binding to antigen; (vi) Molecular modeling and dynamics simulations can identify many significant intermolecular interactions which can be confirmed experimentally. The thermodynamic studies have revealed properties which we believe to be predictive of binding characteristics including long term complex stability and likely cross-reactivity with related antigens, and we are developing a protocol for assessing these properties which would be valuable in selection of lead therapeutic antibodies. The insight(s) gained by analyses of complex kinetics and thermodynamics provide a framework and rationale antibody engineering, informing the design of antibodies of predefined specificity for immunotherapy, and will also lead to better strategies for structure-based drug design and selection of lead compounds in molecular targeting efforts. In addition, the methodology and insight from this project inform design of experiments to study in molecular interaction networks in normal and cancer cells. We have initiated two new structural biology studies to inform our interpretation of structure-activity relationships. We have incorporated fluorinated tryptohan at six tryptophan residues in a single-chain Fv antibody and are using F-19 NMR to study flexibility and conformational changes upon antigen binding. While it often assumed that fluorine labeling proteins does little to perturb the structure, 3 lines of evidence from our data to date indicate that the structure of the labeled protein differs significantly from that of the unlabeled: (1) the CD spectra are significantly different; (2) the binding kinetics and affinties of the the labeled and unlabeled proteins are significantly different (3) shifts in the F-19 spectra strongly suggest that the F-19 alters with association of the heavy and light chains of the Fv. We have begun crystallization trials, in order to compare the structures of both the wild type and the fluorinated ScFv complexes. This would be only the second F-19 incorporated protein in the Protein Data Base, and would yield important information about the conformational effects of F-19 incorporation into proteins. This information is of broad significance because this technique is commonly used for NMR spectroscopy.