Modulation of Dimerization, Binding, Stability, and Folding by Mutation of the Neurophysin Subunit Interface

Abstract

Bovine neurophysins, which have typically served as the paradigm for neurophysin behavior, are metastable in their disulfide-paired folded state and require ligand stabilization for efficient folding from the reduced state. Studies of unliganded porcine neurophysin (oxytocin-associated class) demonstrated that its dimerization constant is more than 90-fold greater than that of the corresponding bovine protein at neutral pH and showed that the increased dimerization constant is accompanied by an increase in stability sufficient to allow efficient folding of the reduced protein in the absence of ligand peptide. Using site-specific mutagenesis of the bovine protein and expression in Escherichia coli, the functional differences between the bovine and porcine proteins were shown to be attributable solely to two subunit interface mutations in the porcine protein, His to Arg at position 80 and Glu to Phe at position 81. Mutation of His-80 alone to Arg had a relatively small impact on dimerization, while mutation to either Glu or Asp markedly reduced dimerization in the unliganded state, albeit with apparent retention of the positive linkage between dimerization and binding. Comparison of the peptide-binding constants of the different mutants additionally indicated that substitution of His-80 led to modifications in binding affinity and specificity that were independent of effects on dimerization. The results demonstrate the importance of the carboxyl domain segment of the subunit interface in modulating neurophysin properties and suggest a specific contribution of the energetics of ligand-induced conformational change in this region to the overall thermodynamics of binding. The potential utility to future studies of the self-folding and monomeric mutants generated by altering the interface is noted.

Protein‐protein interactions as a target for drugs in proteomics

“…Numerous data suggest that the recognition and specificity require the directed forces of interaction such as hydrogen bonds and electrostatic forces, whereas the binding energy is probably also determined by hydrophobic forces [2,19,33,[43][44][45][46][47][48][49][50][51][52][53]. It is also suggested that the most binding energy is related only to several amino acids from the interface, so called "hot spots" [54][55][56][57].…”

Effects of Diabetes Insipidus Mutations on Neurophysin Folding and Function

“…The preparation of recombinant WT mature BNP-I and oxytocin precursor and mutagenesis of wild type cDNA have been described previously (17,20). The recombinant mature mutant proteins in the present study were expressed in Escherichia coli, folded at pH 8.0 in the presence of ␤-mercaptoethanol and 10 mM Phe-Tyr-NH 2 , and purified by affinity chromatography as described for the recombinant mature WT protein (17). A large fraction of the protein so prepared is covalently damaged prior to folding (17,20), and the remaining protein folds with variable efficiency depending on its structure (see below).…”

“…The recombinant mature mutant proteins in the present study were expressed in Escherichia coli, folded at pH 8.0 in the presence of ␤-mercaptoethanol and 10 mM Phe-Tyr-NH 2 , and purified by affinity chromatography as described for the recombinant mature WT protein (17). A large fraction of the protein so prepared is covalently damaged prior to folding (17,20), and the remaining protein folds with variable efficiency depending on its structure (see below). The covalent structures of mutants were confirmed by DNA sequencing of the plasmids from which they were prepared and by mass spectrometry.…”

“…27); the peptides used were Phe-Tyr-NH 2 or Abu-Tyr-NH 2 , depending on availability. Dimerization constants were determined from one-dimensional proton NMR spectra obtained in D 2 O as described previously (17), measuring the intensity ratio at different protein concentrations of signals at ϳ6.4 and 6.2 ppm representing dimer and monomer, respectively; the validity of the use of these signals in the mutants was verified by demonstrating that the calculated dimerization constants were independent of protein concentration.…”

“…The central element of this self-association is the dimer, the formation of which occurs through specific ␤-sheet interactions between folded NP subunits (6,7) and is strongly enhanced by the binding of hormone or related peptides to the binding site (15). The efficiency of NP folding is in turn critically linked to the correct but metastable (16) pairing of its 7 disulfides and depends, with one known exception (17), on the ability of the protein to bind hormone and thereby provide the necessary thermodynamic stability to drive formation of the correct disulfides to completion. Thus, mutations (e.g.…”