NMR investigation of main-chain dynamics of the H80E mutant of bovine neurophysin-I: demonstration of dimerization-induced changes at the hormone-binding site.
Academic Article
Overview
abstract
Neurophysins are hormone-binding proteins composed of two partially homologous domains. Ligand-binding (localized to the amino domain) and dimerization (involves both domains) are cooperatively linked by an as yet undefined allosteric mechanism. To help define this mechanism, we investigated the backbone dynamics of the unliganded monomeric state of the H80E mutant of bovine neurophysin-I by (15)N NMR. Model-free analysis of the NMR relaxation parameters indicated significantly greater flexibility in the carboxyl domain than in the amino domain, particularly at their dimerization interface segments. Amino domain residues critical to hormone binding were highly structured, constraining potential allosteric mechanisms. Model-free analysis additionally demonstrated chemical exchange effects, manifest as R(ex) terms, in 16 residues, 14 of which are located in the amino domain at, or immediately adjacent to, either the dimerization interface or the hormone-binding site. The chemical exchange process was further characterized using relaxation-compensated CPMG measurements, the results allowing assignment of the process to monomer-dimer exchange and calculation of the exchange kinetics, which were slow on the NMR time scale. An apparently different concentration-dependent process, distinguished from normal dimerization by its fast exchange behavior and pH-independence, also principally involved a subset of residues at and immediately adjacent to either the hormone-binding site or the amino domain dimerization interface. The data represent the first direct demonstration of an effect of dimerization in the unliganded state on neurophysin's hormone-binding site, the effect particularly involving residues that interact with hormone residue 2, and specifically identify Ser25 and Ile26 as likely intermediaries between the sites of dimerization and of hormone binding. Consistent with recent views of the role of anchor residues in protein interactions, we propose that dimerization proceeds by a fast pH-independent association of the well-structured amino domain interface that is rapidly communicated to the binding site for hormone residue 2, followed by a rate-determining pH-dependent interaction of the less structured carboxyl domain interface.
