title: Principal determinants leading to transition state formation of a protein-protein complex, orientation trumps side-chain interactions.
abstract: The binding transition state (TS) is the rate-limiting step for transient molecular interactions. This important step in the molecular recognition process, however, is largely understood only at a qualitative level. To establish a more quantitative picture of the TS structure, we exploit a set of biophysical techniques that have provided major insights in protein folding applications. As a model system representing the large class of "weakly charged" protein-protein interactions, we examine the binding of a variety of human growth hormone (hGH) variants to the human growth hormone receptor (hGHR) and the human prolactin receptor (hPRLR). hGH variants were chosen to probe different features of the TS structure, based on their highly reengineered interfaces. Both Eyring and urea (m value) analyses suggest that the majority of binding surface burial occurs after TS. A comprehensive phi analysis showed that individual hGH interface residues do not contribute energetically to the stability of the TS, but there is a TS "hot spot" in the receptor. Zinc dependence studies that take advantage of an endogenous tetracoordinated interfacial metal binding demonstrate that surfaces of the molecules have attained a high orientational complementarity by the time the TS is reached. The model that best fits these data are that a "knobs-into-holes" process precisely aligns the two molecular interfaces in forming the TS structure. Surprisingly, most of the thermodynamic character of the binding reaction is focused in the fine-tuning process occurring after TS.
authors.affiliation: Department of Biochemistry and Molecular Biology, and Institute for Biophysical Dynamics, and Computation Institute, University of Chicago, Chicago, IL 60637, USA.
authors.forename: James R
authors.initials: JR
authors.lastname: Horn
authors.forename: Tobin R
authors.initials: TR
authors.lastname: Sosnick
authors.forename: Anthony A
authors.initials: AA
authors.lastname: Kossiakoff
mesh_terms: D019382:Human Growth Hormone / Q000737:chemistry* / Q000378:metabolism; D008565:Membrane Proteins / Q000378:metabolism*; D008958:Models, Molecular; D011485:Protein Binding; D020349:Surface Plasmon Resonance; D013816:Thermodynamics
chemical_list: D008565:Membrane Proteins; C405010:delta-hGHR; D019382:Human Growth Hormone