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, a conclusion recently confirmed by solution experiments using fluorescence spectroscopy; (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, than affinity per se, 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; (vii) Water activity (studied by osmotic pressure experiments) is critical in understanding the thermodynamics of antibody-antigen association. Recently, using refolded Fab chain-chimeras in a family of structurally and functionally related antibodies, we characterized a series somatic light-chain mutations thermodynamically for their impact on antigen binding affinity. The results have revealed one possible strategy for engineering enhanced affinity in high affinity antibodies by mutating to glycine non-contact residues adjacent to residues binding hotspots on the antigen. We recently obtained a high resolution crystal structure of the Fab10 complex (1.2, pdb 3D9A), in collaboration with A. Wlodawer (LMC, CCR), which contains the glycine substitution and exhibits an increased complementarity for antigen of the antibody over the surface surrounding the substitution, in comparison with structures of Fab26 and Fab63 complexes which do not contain the glycine substitution. This hypothesis has been tested by production of a large number of site directed mutants and kinetic analyses of expressed and refolded mutated proteins. These experiments are largely complete; some double- and triple-mutant cycle experiments in progress, with parallel molecular dynamics simulations, to test the hypothesis that size and flexibility, rather than hydrophobicity, is the underlying mechanism of action of the introduced mutations. Also in progress are experiments utilizing DSC and fluoresence quenching to better understand time-dependent kinetics, a model which we have developed utilizing SPR results and are developing for examination of other receptor-ligand complexes. 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 5-19F-tryptohan (5FW) at six tryptophan residues in a single-chain Fv of the antibody HyHEL-10 (scFv10), and are using 19F NMR (in collaboration with J. Barchi, LMC, CCR) to study flexibility and conformational changes upon antigen binding. In order to identify the 19F resonance peaks, site directed mutants were made for each Trp residue, and each of these had their own unique problems for expression and folding. Information from T2 relaxation times during binding is being analysed to better understand changes in flexibility & dynamics at and around each of six fluoro probe position. The results to date unambiguously show a significant induced fit upon binding, even though previous comparisons by X-ray crystallography of a closely related antibody complexed and uncomplexed did not indicate large structural changes upon binding. In addition, shifts in the 19F spectra strongly suggest that the 19F alters with association of the heavy and light chains of the scFv10. While it is often assumed that fluorine labeling proteins does little to perturb the structure, many lines of evidence (including binding kinetics and affinities, CD spectra, and fluorescence titrations) indicate that the structure of the 5FW labeled scFv10 differs significantly from that of the unlabeled, and that the structures of protein labeled with 5FW and 6FW differ from each other. Therefore, M.E. DeSantis from my laboratory, in collaboration with the laboratory of Dr. Wlodawer, crystallized the complexes of both the wild type and the fluorinated ScFv10 complexes with HEL in order to compare their structures (pdb 2ZNW and 2ZNX, to be published). The crystal structures of the 5FW and unlabeled complexes do not show significant structural differences, consistent with the findings of the single other 19F-incorporated protein in the Protein Data Base. We hypothesize that complexation is accompanied by a strong induced fit to the relatively rigid lysozyme, which also seeds the crystals, obscuring structural or dynamic differences which may be important in solution. We collected solution Small Angle X-ray Scattering (SAXS) data on 5FW-labeled and unlabeled scFv10 both complexed and uncomplexed with antigen in collaboration with the laboratory of Y-X Wang (SBL, CCR). Preliminary analyses suggest significant differences between the labeled and unlabeled proteins, and that there may be more difference between the uncomplexed proteins, supporting our hypothesis of a strong induced fit upon complex formation. This information is of broad significance because this technique is commonly used for NMR spectroscopy.

National Institute of Health (NIH)
National Cancer Institute (NCI)
Intramural Research (Z01)
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National Cancer Institute Division of Basic Sciences
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Li, Yili; Huang, Yuping; Swaminathan, Chittoor P et al. (2005) Magnitude of the hydrophobic effect at central versus peripheral sites in protein-protein interfaces. Structure 13:297-307
Sinha, Neeti; Smith-Gill, Sandra J (2005) Molecular dynamics simulation of a high-affinity antibody-protein complex: the binding site is a mosaic of locally flexible and preorganized rigid regions. Cell Biochem Biophys 43:253-73
Stroh, Cordula M; Ebner, Andreas; Geretschlager, Manfred et al. (2004) Simultaneous topography and recognition imaging using force microscopy. Biophys J 87:1981-90
Li, Yili; Li, Hongmin; Yang, Feng et al. (2003) X-ray snapshots of the maturation of an antibody response to a protein antigen. Nat Struct Biol 10:482-8