STRUCTURAL AND KINETIC ANALYSIS OF CD4 MUTANTS THAT ARE DEFECTIVE IN HIV BINDING. Hao Wu¹, David G. Myszka², Susan W. Tendian³, Christie G. Brouillette³, Ray W. Sweet², Irwin M. Chaiken², Wayne A. Hendrickson^1,2, ¹Department of Biochemistry and Molecular Biophysics, Columbia University, 630 West 168th Street, New York, NY 10032, ²SmithKline Beecham Pharmaceuticals, King of Prussia, PA, 19406, ³Southern Research Institute, Birmingham, Alabama, 35205

It is well established by equilibrium binding studies that CD4 interacts with gpl20 in the range of nanomolar affinity. Little is known, however, about the mechanism of this interaction. In this report, we analyzed the native and several mutant forms of the HIV-binding fragment (D1D2) of CD4 using a combination of kinetic, structural and thermodynamic approach. Our real-time binding kinetic data from BIAcore measurements showed that the affinity decrease in HIV-binding defective mutants is mainly due to the decrease in association rate. The predominant alteration of association-rate by neutral mutations may invariantly suggest conformational adaptation in this and many other protein-protein interactions.