MODULATION OF ALLOSTERIC INTERACTIONS IN NEUROPHYSIN INDUCED BY SUCCINYLATION OF SERINE-56 OR CLEAVAGE OF RESIDUES-1-8

Neurophysin is an allosteric protein in which peptide binding and self -association are positively linked. Reaction of neurophysin with succi nic anhydride led to a large decrease in peptide affinity assignable t o succinylation of a serine or threonine hydroxyl group. To identify t he residue involved, acetimidated protein was reacted with [C-14]succi nic anhydride and the active and inactive components were separated by affinity chromatography. Performic acid oxidation and tryptic and Asp -N mapping of the two components, followed by automated Edman degradat ion, allowed identification of the critical residue as Ser-56. This re sidue is not a direct participant in peptide binding and is distant fr om the subunit interface of the dimer, but it is immediately adjacent to the site of one of the known mutations associated with familial dia betes insipidus. Examination in solution of the peptide affinity of ne urophysin succinylated at Ser-56 indicated a binding affinity approxim ately 1/20th that of the native protein or of protein succinylated at other residues, and a loss of the normal dependence of binding affinit y on protein concentration. Under the same buffer conditions, loss of the concentration dependence of binding, in addition to the previously demonstrated loss of binding affinity, also accompanied excision of r esidues 1-8, an effect attributed to the loss of binding site residue Arg-8. However, in contrast to the effects of succinylation on native neurophysin, only minor effects of succinylation on the binding affini ty of the des-1-8 protein were observed. The results indicate that the effects of succinylation and of cleavage of the 1-8 sequence on bindi ng affinity are mediated by a common mechanism that additionally alter s self-association under appropriate ionic conditions. Examination of the neurophysin crystal structure indicates a distance of <8 angstrom between Arg-8 and Ser-56, suggesting that the two residues are part of a common structural locus involved in long-range interactions between the binding site and the subunit interface.