Site2 binding energetics of the regulatory step of growth hormone-induced receptor homodimerization.

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TitleSite2 binding energetics of the regulatory step of growth hormone-induced receptor homodimerization.
Publication TypeJournal Article
Year of Publication2003
AuthorsWalsh, STR, Jevitts, LM, Sylvester, JE, Kossiakoff, AA
JournalProtein Sci
Volume12
Issue9
Pagination1960-70
Date Published2003 Sep
ISSN0961-8368
KeywordsAlanine, Binding Sites, Dimerization, Epitopes, Human Growth Hormone, Humans, Kinetics, Models, Molecular, Mutation, Protein Binding, Protein Structure, Secondary, Protein Structure, Tertiary, Surface Plasmon Resonance, Time Factors
Abstract

Receptor signaling in the growth hormone (GH)-growth hormone receptor (GHR) system is controlled through a sequential two-step hormone-induced dimerization of two copies of the extracellular domain (ECD) of the receptor. The regulatory step of this process is the binding of the second ECD (ECD2) to the stable preassociated 1 : 1 GH/ECD1 complex on the cell surface. To determine the energetics that governs this step, the binding kinetics of 38 single- and double-alanine mutants in the hGH Site2 contact with ECD2 were measured by using trimolecular surface plasmon resonance (TM-SPR). We find that the Site2 interface of hGH does not have a distinct binding hot-spot region, and the most important residues are not spatially clustered, but rather are distributed over the whole binding surface. In addition, it was determined through analysis of a set of pairwise double alanine mutations that there is a significant degree of negative cooperativity among Site2 residues. Residues that show little effect or even improved binding on substitution with alanine, when paired with D116A-hGH, display significant negative cooperativity. Because most of these pairwise mutated residues are spatially separated by >or=10 A, this indicates that the Site2 binding interface of the hGH-hGHR ternary complex displays both structural and energetic malleability.

DOI10.1110/ps.03133903
Alternate JournalProtein Sci.
PubMed ID12930995
PubMed Central IDPMC2323993
Grant ListDK-61602 / DK / NIDDK NIH HHS / United States