Bovine neurophysin dimerization and neurohypophyseal hormone binding

Nicolas, Pierre; Batelier, Gérard; Rholam, Mohamed; Cohen, Paul A.

doi:10.1021/bi00556a023

Cited by 64 publications

(93 citation statements)

References 51 publications

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“…The monomer-monomer interface of dimer II is additionally stabilized by the fifth dipeptide. This extra site may correspond to the minor binding site reported for small peptides (33) and hormones (32). The a-NH' of this peptide is bonded to the carboxyl groups of the two Glu' residues from the constituent monomers 3 and 4.…”

Section: Resultsmentioning

confidence: 66%

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Crystal structure of a bovine neurophysin II dipeptide complex at 2.8 A determined from the single-wavelength anomalous scattering signal of an incorporated iodine atom.

Chen

Rose

Breslow

et al. 1991

Proc. Natl. Acad. Sci. U.S.A.

142

144

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The crystal structure of a dipeptide complex of bovine neurophysin H has been solved at 2.8 A resolutionsolely by using single-wavelength anomalous scattering data from a single iodinated derivative. The asymmetric unit is an elongated tetramer of dimensions 110 x 40 X 30 A, composed of two dimers related by pseudo twofold symmetry. Each monomer consists of two homologous layers, each with four antiparaflel 3-strands. The two regions are connected by a helix followed by a long loop. Monomer-monomer contacts involve antiparallel 13-sheet interactions, which form a dimer with two layers of eight P-strands. (10), one of which is isomorphous with the crystals of a porcine NP-I complex (11). These contained 4, 8, and 12 molecules per asymmetric unit, respectively, suggesting that the basic aggregates of the NP-dipeptide complex in the crystals are tetramers that can self-associate to higher oligomers (10). In view of the probable importance of NP self-association in packaging of the precursor in the NSG (1, 4) and evidence that NP complexes can be present in the NSG as the precipitated or crystalline state (12, 13), analysis of the crystal structure of these complexes offers an opportunity for providing detailed structural information not only on NP folding and protein-peptide interactions but also on how the complexes might be packaged in NSG.The crystal structure of a bovine NP-II-dipeptide complex"l reported here was determined using only the singlewavelength anomalous scattering (SAS) data from an iodinated derivative crystal. Iodine was chosen as a probe because of its relatively large anomalous scattering signal (Alf' = 6.8) and its ease of incorporation into the dipeptide.The resulting derivative crystals contained iodine atoms covalently linked to the Phe residues of the bound hormone analogs.Here we report the structure of a NP molecule, as well as aspects of the methodology used in solving the structure. METHODSCocrystaflization. Bovine NP-I was purified as described (14). The dipeptide para-iodo-L-phenylalanyltyrosine amide (I-Phe-Tyr-NH2) was custom-synthesized by Peninsula Laboratories and was demonstrated by using circular dichroism (8) to bind to the hormone-binding site with high affinity. Crystals of the NP-dipeptide complex were grown at pH 7.5 by using a modification of the procedure of Yoo et al. (9); crystals of the complex of the corresponding noniodinated peptide served as seeds. The crystals obtained were nearly isomorphous with those of the complex of the uniodinated peptide, having four molecules (chains) per asymmetric unit and space group P212121 with a = 120.0 A, b = 69.4 A, and c = 62.4 A.

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Section: Resultsmentioning

confidence: 66%

“…NPs form dimers in solution, and dimerization is facilitated by the binding of hormone or analogous small peptides (32). The overall dimensions of the NP dimers are 68 x 32 x 30 A, differing from the spherical shape calculated from hydrodynamic data (4).…”

Section: Resultsmentioning

confidence: 97%

Crystal structure of a bovine neurophysin II dipeptide complex at 2.8 A determined from the single-wavelength anomalous scattering signal of an incorporated iodine atom.

Chen

Rose

Breslow

et al. 1991

Proc. Natl. Acad. Sci. U.S.A.

142

144

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“…Leakage through a damaged granule membrane would, at first sight, be expected to produce greater loss of hormone than of neurophysin on the basis of molecular size. However, consideration of the Stokes radius of neurophysin molecules indicates that monomeric, but not dimeric (16), neurophysin could diffuse out of smooth membrane-limited vesicles (17). Therefore, aging of NSG could include a change from dimeric to monomeric neurophysin.…”

Section: Discussionmentioning

confidence: 99%

Method for quantitating the molecular content of a subcellular organelle: hormone and neurophysin content of newly formed and aged neurosecretory granules.

Nordmann¹,

Morris²

1984

Proc. Natl. Acad. Sci. U.S.A.

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A method is described for the quantitative determination of the content of subcellular organelles such as secretory granules. Purified subcellular fractions of the organelle are prepared and aliquots are assayed for hormones, for example. To determine the number of organelles per fraction, known numbers of latex particles of a size similar to the organelle are added to other aliquots of the subcellular fractions. Latex particles and organelles are then pelleted together by centrifugation. The ratio between latex particles and organelles can be determined by morphometric analysis of ultrathin sections taken through the full thickness of the pellet. The number of organelles and hence their content of the substance assayed can then be calculated. We have applied this technique to posterior pituitary neurosecretory granules, the content of which has already been estimated by a different method. Newly formed neurosecretory granules from oxen and rats were found to have a content of -85,000 molecules of hormone and neurophysin. Aged neurosecretory granules from the same neural lobes appeared to contain less hormone and neurophysin, but this was shown to be the result of loss of material from the granules during isolation in media of 360 mosM. Such loss could be prevented by isolation in hypertonic (660 mosM) media.

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“…Residual native protein was separated from the cleaved protein by affinity chromatography (15), the cleaved protein not binding to the affinity column. On native polyacrylamide gel electrophoresis at pH 9.5, the non-binding protein gave two bands, a major product (Ն80% of the protein) migrating with the expected additional negative charge relative to the native protein and a minor band with the same mobility as the native protein.…”

Section: Preparation Of Wild Type and Recombinant Proteins-bnp-imentioning

confidence: 99%

“…In the case of oxytocin and vasopressin precursors, the self-association arises from the self-association properties of their NP segments. 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.…”

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confidence: 99%

Effects of Diabetes Insipidus Mutations on Neurophysin Folding and Function

Eubanks¹,

Nguyen²,

Deeb³

et al. 2001

Journal of Biological Chemistry

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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...

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Bovine neurophysin dimerization and neurohypophyseal hormone binding

Cited by 64 publications

References 51 publications

Crystal structure of a bovine neurophysin II dipeptide complex at 2.8 A determined from the single-wavelength anomalous scattering signal of an incorporated iodine atom.

Crystal structure of a bovine neurophysin II dipeptide complex at 2.8 A determined from the single-wavelength anomalous scattering signal of an incorporated iodine atom.

Method for quantitating the molecular content of a subcellular organelle: hormone and neurophysin content of newly formed and aged neurosecretory granules.

Effects of Diabetes Insipidus Mutations on Neurophysin Folding and Function

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