Modulation of dimerization, binding, stability, and folding by mutation ofthe neurophysin subunit interface

Bovine neurophysins, which have typically served as the paradigm for neurop hysin behavior, are metastable in their disulfide-paired folded state and r equire ligand stabilization for efficient folding from the reduced state. S tudies of unliganded porcine neurophysin (oxytocin-associated class) demons trated that its dimerization constant is more than 90-fold greater than tha t of the corresponding bovine protein at neutral pH and showed that the inc reased dimerization constant is accompanied by an increase in stability suf ficient 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 difference's. between the bo vine and porcine proteins were shown to be attributable solely to two subun it interface mutations in the porcine protein, His to Arg at position 80 an d Glu to Phe at position 81. Mutation of His-80 alone to Arg had a relative ly small impact on dimerization, while mutation to either Glu or Asp marked ly reduced dimerization in the unliganded state, albeit with apparent reten tion of the positive linkage between dimerization and binding. Comparison o f the peptide-binding constants of the different mutants additionally indic ated that substitution of His-80 led to modifications in binding affinity a nd specificity that were independent of effects on dimerization. The result s demonstrate the importance of the carboxyl domain segment of the subunit interface in modulating neurophysin properties and suggest a specific contr ibution of the energetics of ligand-induced conformational change in this r egion to the overall thermodynamics of binding. The potential utility to fu ture studies of the self-folding and monomeric mutants generated by alterin g the interface is noted.