Mechanisms underlying the pathogenicity of diabetes insipidus mutations were probed by studying their effects on the properties of bovine oxytocin-related neurophysin. The mutations G17V, ⌬E47, G57S, G57R, and C67STOP were each shown to have structural consequences that would diminish the conformational stability and folding efficiency of the precursors in which they were incorporated, and factors contributing to the origins of these property changes were identified. Effects of the mutations on dimerization of the folded proteins were similarly analyzed. The projected relative impact of the above mutations on precursor folding properties qualitatively parallels the reported relative severity of their effects on the biological handling of the human vasopressin precursor, but quantitative differences between thermodynamic effects and biological impact are noted and explored. The sole mutation for which no clear thermodynamic basis was found for its pathogenicity was 87STOP, suggesting that the region of the precursor deleted by this mutation plays a role in targeting independent from effects on folding, or participates in stabilizing interactions unique to the human vasopressin precursor.The hormone vasopressin is synthesized as part of a larger precursor that contains the vasopressin sequence at its amino terminus, followed by that of the disulfide-rich protein neurophysin (NP) 1 and a carboxyl-terminal glycopeptide known as copeptin (Ref. 1 and reviewed in Ref. 2). Oxytocin biosynthesis is similar (3), with the exception that the precursor lacks the copeptin segment, the function of which is unclear (4). The NP components of the two precursors ( VP NP) and ( Oxy NP) are highly homologous; the two bovine neurophysins have almost identical properties in vitro, including similar affinities for each of the two hormones (e.g. Ref.2). Following processing, which cleaves the hormones from NP, the mature hormones remain noncovalently bound to NP by forces basically analogous to those pre-existing within the precursor, leading to analogous NP self-association properties (5). The structure of NP, the nature of the noncovalent interactions between hormones and NP, and the effects of these interactions on NP properties have been extensively investigated in solution (e.g. Ref.2) and by crystallographic analysis (6, 7). Moreover, the central role of NP in vasopressin elaboration has become evident by the demonstration that familial neurogenic diabetes insipidus (FNDI), an autosomal dominant disease characterized by vasopressin deficiency, appears most frequently to be due to mutations in NP (e.g. Refs. 8 and 9) accompanied by loss of the proper targeting of the hormone to regulated neurosecretory granules (e.g. Refs. 10 and 11); retention of the mutated precursor in the endoplasmic reticulum is generally considered the cause of the ultimate death of the affected neurons (10).Many of the mutations involved in diabetes insipidus can be predicted, on the basis of what is already known, to exert their effects by directly or indir...
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.
Earlier thermodynamic studies of the intermolecular interactions between mature oxytocin and neurophysin, and of the effects of these interactions on neurophysin folding, raised questions about the intramolecular interactions of oxytocin with neurophysin within their common precursor. To address this issue, the disulfide-rich precursor of oxytocin-associated bovine neurophysin was expressed in Escherichia coli and folded in vitro to yield milligram quantities of purified protein; evidence of significant impediments to yield resulting from damage to Cys residues is presented. The inefficiency associated with the refolding of reduced mature neurophysin in the presence of oxytocin was found not to be alleviated in the precursor. Consistent with this, the effects of pH on the spectroscopic properties of the precursor and on the relative stabilities of the precursor and mature neurophysin to guanidine denaturation indicated that noncovalent intramolecular bonding between oxytocin and neurophysin in the precursor had only a small thermodynamic advantage over the corresponding bonding in the intermolecular complex. Loss of the principal interactions between hormone and protein, and of the enhanced stability of the precursor relative to that of the mature unliganded protein, occurred reversibly upon increasing the pH, with a midpoint at pH 10. Correlation of these results with evidence from NMR studies of structural differences between the precursor and the intermolecular complex, which persist beyond the pH 10 transition, suggests that the covalent attachment of the hormone in the precursor necessitates a conformational change in its neurophysin segment and leads to properties of the system that are distinct from those of either the liganded or unliganded mature protein.
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