Our Science – Smith-Gill Website

Sandra J. Smith-Gill, Ph.D.

Structural Biophysics Laboratory Head, Structural Immunology Section Senior Investigator NCI-Frederick Structural Immunology Section Building 538, Room 140 Frederick, MD 21702-1201 Phone:   301-846-5203 Fax:   301-846-6326 E-Mail:   smithgil@helix.nih.gov

Biography

Dr. Sandra Smith-Gill obtained her Ph.D. in developmental biology and genetics from the University of Michigan. She has been on the faculties of Swarthmore College, George Washington University, and the University of Maryland. She joined the National Cancer Institute in 1981, and until 1998 was in the Laboratory of Genetics in Bethesda, where her laboratory worked on mechanisms of plasmacytoma induction in a mouse model, and structure-function relationships in antibodies. Her group was among the first to map epitopes on a defined protein antigen using monoclonal antibodies, and contributed seminal work towards our current view of protein antigenic structure. In 1998 she joined the Basic Research Laboratory, CCR, NCI-Frederick, and in 1992 she established the Structural Immunology Group within the Structural Biophysics Laboratory. The SIS group investigates molecular recognition to elucidate the general principles of protein target recognition by receptors, using an interdisciplinary approach. 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.

Research

Structural and Functional Analysis of Molecular Recognition in Receptor-Ligand Protein-Protein Complexes

Macromolecular interactions are central to cellular regulation and biological function. The Structural Immunology Section investigates molecular recognition in antibody complexes with proteins. Several antibody-protein complexes have been studied in depth as model systems to elucidate the general principles of protein-protein interactions. Antibodies complexed with hen egg-white lysozyme (HEL) are among the most thoroughly studied, including several originating from our laboratory. We were the first to map the protein antigenic epitope recognized by a monoclonal antibody, and to publish evidence that the entire surface of a protein is potentially antigenic. This finding challenged and completely changed the then-current paradigm that only limited, predefined sites on a protein were antigenic and the response was host-specific, the paradigm which still holds today and continues to be an important and critical consideration in understanding the immune response to a particular drug or treatment, vaccine development and antibody design.

Antibody Affinity Maturation. The humoral immune response is unique because it includes a large repertoire of antibodies encompassing a broad range of affinities and specificities, including both multi-and poly-reactive antibodies. During maturation of the antibody response, the somatic mutations are introduced into the antibody variable regions, and clonal selection leads to expansion of B-cell clones with higher affinity binding. During this process, referred to as affinity maturation, average affinity typically increases by 10-100 fold, and the antibody response also becomes more specific. Antibody affinity maturation thus represents a prototypical example of molecular evolution. X-ray snapshots of mAbs isolated at different times during the evolution of the response to HEL provided the first structural view of maturation of the antibody response to a protein antigen, revealing that in contrast to anti-hapten antibodies, higher affinity binding does not correlate with increases in numbers of contacts, hydrogen bonds, or buried surface area per se, but rather with increased complementarity and burial of apolar surface which is increased at the expense of polar surface.

Kinetics & Thermodynamics of Antibody-Antigen Protein-Protein Association. We have developed novel protocols for surface plasmon resonance (SPR) analysis which have provided important evidence that (i) Antibody-antigen bimolecular association is a time-dependent 2-step binding process, a conclusion recently confirmed by solution experiments using fluorescence spectroscopy. As a consequence, efficacy of an inhibitor can also be time-dependent; (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 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) Water activity (studied by osmotic pressure experiments) is critical in understanding the thermodynamics of antibody-antigen association. These studies provide new insights on the mechanism of antibody-antigen binding, and in addition have provided new methodology for examining these interactions.

Conformational Flexibility & Induced Fit. Although association of many receptor-ligand complexes is accompanied by conformational rearrangements, the role of these rearrangements or 'induced fit' in high affinity antibody-ligand complexes is not completely understood. Using refolded Fab chain-chimeras, we characterized a series somatic light-chain mutations thermodynamically for their impact on antigen binding affinity. We recently obtained a high resolution crystal structure of the Fab10 complex (1.2A, pdb 3D9A) which contains the glycine substitution and exhibits an increased complementarity for antigen of the antibody over the surface surrounding the substitution This hypothesis has been tested by introduction of glycine mutations into comparable positions at the interface to look for similar patterns on the effects of binding. Computational studies using molecular dynamics are also underway probing the relative changes in flexibility of the CDR loops containing this mutation.

We hypothesize that complexation is accompanied by a strong induced fit to the relatively rigid lysozyme. 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- to study flexibility and conformational changes upon antigen binding. Changes in chemical shift and measurement of T2 relaxation parameters allows us to characterize specific changes occurring at each of those six positions during binding to the natural and mutated HEL In addition, shifts in the 19F spectra strongly suggest that the 19F alters with association of the heavy and light chains of the scFv10. X-ray crystallography and Small Angle X-Ray Scattering data are being analyses to gain additional insight about the solution structures of these complexes.

Collaborators: We have collaborated in this research with Roy Mariuzza, Center for Advanced Research in Biotechnology, University of Maryland; Yung.-Xing Wang, SBL, CCR; Joe Barchi, LMC, CCR; Alex Wlodowar, MCL, CCR; and Richard Willson, University of Houston.

This page was last updated on 7/31/2008.