Esther Breslow

Crystal structure of the neurophysin—oxytocin complex

The first crystal structure of the pituitary hormone oxytocin complexed with its carrier protein neurophysin has been determined and refined to 3.0 Å resolution. The hormone-binding site is located at the end of a 310-helix and involves residues from both domains of each monomer. Hormone residues Tyr 2, which is buried deep in the binding pocket, and Cys 1 have been confirmed as the key residues involved in neurophysin-hormone recognition. We have compared the bound oxytocin observed in the neurophysin–oxytocin complex, the X-ray structures of unbound oxytocin analogues and the NMR-derived structure for bound oxytocin. We find that while our structure is in agreement with the previous crystallographic findings, it differs from the NMR result with regard to how Tyr 2 of the hormone is recognized by neurophysin.

Physical-chemical properties of ubiquitin.

The secondary structure of ubiquitin, the environment of its single tyrosine residue and its potential for interacting noncovalently with histone 2A or DNA, have been probed by circular dichroism (CD), ultraviolet absorbance, fluorescence and ancillary techniques. The results indicate that ubiquitin has a stable secondary structure containing only a low percentage of alpha-helix or beta-sheet. The ubiquitin tyrosine has an elevated pKa arising from the influence of a spatially proximate carboxylate which also causes a marked quenching of the tyrosine fluorescence at neutral pH; the influence of this carboxylate is lost when the protein is unfolded in 7 M guanidine. No evidence has been obtained for the presence of allosteric noncovalent interactions between free ubiquitin and either histone 2A or purified unfractionated DNA. The results suggest that one function of ubiquitin (or of the ubiquitin segment of protein A24) may be to interact with a chromatin component other than histone 2A or DNA, and/or that ubiquitin functions within 2A as a steric blocking group of a region of the nucleosome.

Structures of an unliganded neurophysin and its vasopressin complex: implications for binding and allosteric mechanisms.

The structures of des 1–6 bovine neurophysin‐II in the unliganded state and as its complex with lysine vasopressin were determined crystallographically at resolutions of 2.4 Å and 2.3 Å, respectively. The structure of the protein component of the vasopressin complex was, with some local differences, similar to that determined earlier of the full‐length protein complexed with oxytocin, but relatively large differences, probably intrinsic to the hormones, were observed between the structures of bound oxytocin and bound vasopressin at Gln 4. The structure of the unliganded protein is the first structure of an unliganded neurophysin. Comparison with the liganded state indicated significant binding‐induced conformational changes that were the largest in the loop region comprising residues 50–58 and in the 7–10 region. A subtle binding‐induced tightening of the subunit interface of the dimer also was shown, consistent with a role for interface changes in neurophysin allosteric mechanism, but one that is probably not predominant. Interface changes are suggested to be communicated from the binding site through the strands of β‐sheet that connect these two regions, in part with mediation by Gly 23. Comparison of unliganded and liganded states additionally reveals that the binding site for the hormone α‐amino group is largely preformed and accessible in the unliganded state, suggesting that it represents the initial site of hormone protein recognition. The potential molecular basis for its thermodynamic contribution to binding is discussed.

Nuclear magnetic resonance spin label studies of neurophysin: Evidence for secondary peptide-binding sites☆

Abstract The longitudinal relaxation time, T 1 , of the ortho ring protons of Tyr-49 of bovine neurophysin-I was investigated in the absence and presence of two spinlabeled peptides which are known to bind to the principal hormone-binding site. Displacement of the spin labels from the principal hormone-binding site by diamagnetic peptide led to anomalous changes in T 1 . The results suggest the presence of a second binding site for peptides, of low thermodynamic affinity, but immediately adjacent to Tyr-49.

The Amino Acid Sequence of Rat Kidney 5-Oxo-L-Prolinase Determined by cDNA Cloning

5-Oxoprolinase (EC 3.5.2) catalyzes a reaction in which the endergonic cleavage of 5-oxo-L-proline to form L-glutamate is coupled to the exergonic hydrolysis of ATP to ADP and inorganic phosphate. Highly purified preparations of the enzyme have been obtained from rat kidney and Pseudomonas putida. The rat kidney enzyme is composed of two strongly interacting, apparently identical subunits (Mr = 142,000), whereas that from P. putida is composed of two functionally different protein components that can readily be dissociated. Here we report the cloning of rat kidney 5-oxoprolinase with preliminary expression studies. cDNA clones encoding the enzyme were isolated by screening a λgt11 cDNA library beginning with a degenerate oligonucleotide probe based on peptide sequence data obtained from the purified enzyme. The whole cDNA clone was completed by amplifying its 5′ end from a premade library of rat kidney Marathon-Ready™ cDNAs using polymerase chain reaction methodology. The composite cDNA (4,016 bases) revealed an uninterrupted open reading frame encoding 1,288 amino acid residues (Mr = 137,759). The deduced amino acid sequence contains all four of the peptide sequences that were independently found in peptide fragments derived from the enzyme. Expression of the full-length clone in Escherichia coli yielded a product of the same size as the rat kidney enzyme and which reacted with antibodies directed against the rat kidney enzyme. The predicted amino acid sequence is almost 50% identical throughout its entire length to that of a hypothetical yeast protein YKL215C. It is also 26% identical in half its length to the bacterial hydantoinase HyuA and 26% identical in the other half to the bacterial hydantoinase HyuB. The results suggest unexpected evolutionary relationships among the hydantoinases and rat kidney 5-oxoprolinase which share the common property of hydrolyzing the imide bond of 5-membered rings but which do not all require ATP.

Chemical modification or excision of neurophysin arginine-8 is associated with loss of peptide-binding ability☆

Abstract The role of neurophysin arginines in the binding of peptides to the principal hormone-binding site was evaluated by modification of neurophysin with cyclohexanedione and by limited proteolysis with trypsin. Binding affinities were reduced to values less than 3% of normal when both Arg-8 and Arg-20 were chemically modified or when residues 1–8 were excised by trypsin. Because residues 1–6 are non-essential to binding and residue 7 is not conserved in evolution, the results are interpreted in terms of a functional role for Arg-8.

Methods of Isolation and Identification of Neurophysin Proteins

According to the theory of neurosection proposed by Bargmann and Scharrer (1951), neurohypophyseal hormones are synthesized in hypothalamic nerve cell bodies and carried by axoplasmic transport to nerve terminals in the posterior pituitary, where they are stored and released. The hormones are found in association with higher molecular weight proteins, the neurophysins (Fig. 1).

Crystals of a bovine neurophysin II-dipeptide amide complex☆

Bovine neurophysin II, a hormone carrier protein, has been crystallized as a binary complex with l-phenylalanyl-l-tyrosine amide, a peptide known to bind neurophysin at its hormone binding site. The crystals belong to space group P212121 with cell constants of a = 121.6(10) A, b = 67·9(6) A and c = 62·1(6) A. The density of the crystal is 1.156 g/cm3. There is a tetramer or two dimers in an asymmetric unit.

Inhibition of ubiquitin-dependent proteolysis by des-Gly-Gly-ubiquitin: Implications for the mechanism of polyubiquitin synthesis

Cleavage of the two carboxyl-terminal glycine residues from native ubiquitin yields the proteolysis-incompetent derivative des-Gly-Gly-ubiquitin. We report here that this derivative inhibits the ATP-dependent degradation of casein and is multi-ubiquitinated but not degraded by reticulocyte lysates. Inhibition of proteolysis diminished with increasing concentration of native ubiquitin, but was not reduced by increased casein concentration. Cleavage of the last four residues from ubiquitin yielded a derivative that was a weaker inhibitor of proteolysis and a poorer substrate for ubiquitination. These results suggest that the conjugation of ubiquitin to ubiquitin during polyubiquitin synthesis involves a specific conjugation system that recognizes ubiquitin and some of its derivatives, but not general proteolysis substrates, as ubiquitin acceptors.

Application of high-performance liquid chromatography in neurophysin disulfide assignment

The combined use of ion-exchange, and reversed-phase high-performance liquid chromatography (HPLC) for the isolation of cystine-containing peptides from highly heterogeneous products of the proteolytic digestion of bovine neurophysins is described. The protein was sequentially cleaved by enzymes of decreasing specificity; the peptides released were initially fractionated by gel chromatography and then purified by HPLC. The purified peptides were analyzed by determination of their amino acid composition and mass spectrometry, supported by sequencing techniques. Three of the seven disulfide pairs of neurophysin have now been assigned. The usefulness of the combined use of HPLC and mass spectrometry in assigning these and the other disulfide pairs is illustrated.