Transfer nuclear Overhauser effect study of the conformation of oxytocin bound to bovine neurophysin I

Abstract

This study reports the structure of the peptide hormone oxytocin bound to its carrier protein, neurophysin I, obtained by nuclear magnetic resonance techniques. At the pH value of 2.1 in our experiments, the ligand is in fast exchange with its carrier protein, allowing the use of transfer-NOE methods. The number of distance constraints for the peptide being limited, considerable attention has been paid to an accurate distance determination. The resulting accurate distance limits were used as input for a distance geometry calculation followed by a restrained molecular dynamics run. Convergence to a well-defined family of structures for oxytocin in its bound state was reached. Both the backbone and the side-chain conformations differ between the bound form and the crystal structure of free oxytocin [Wood, S. P., et al. (1986) Science 232, 633]. These differences, as well as other structural features of the bound form, are discussed in terms of interactions made with the carrier protein. Transfer-NOE experiments at low peptide protein ratios provide direct experimental evidence for contacts between the oxytocin Tyr2 residue and an aromatic residue of neurophysin. The resonance assignments of the aromatic groups [Whittaker, B. A., et al. (1985) Biochemistry 24, 2782] together with the recently published X-ray structure of the neurophysin II protein complexed with a dipeptide [Chen et al. (1991) Proc. Natl. Acad. Sci. U.S.A. 88, 4240] allow us to assign the aromatic signal on the protein to the neurophysin Phe22 residue.

Selenoether oxytocin analogues have analgesic properties in a mouse model of chronic abdominal pain

“…The backbone chemical shifts of residues 7-9 are consistent with random coil values and were predicted to be dynamic by Talos þ 41 , in agreement with previous studies (region coloured in red in Fig. 5b) 38 . The unfavourable NMR properties of sulphur make disulphide bonds largely 'invisible' in NMR studies 42 ; in contrast, the SeCtt analogue provided structural constraints through 77 Se couplings to neighbouring methylenes while the additional methylene within the hydrophobic core 14 generated extra distance restraints.…”

“…Later, NMR studies of OT were focused on its conformation when bound to neurophysin 37 . This latter study reported a lack of inter-residue dipolar connectivities in the absence of neurophysin, and subsequent studies have therefore assumed that OT is flexible and unstructured in solution 38 . This has led to the suggestion that binding to the OT receptor may occur through a receptor-induced fit 34 .…”

“…The use of ultra-high magnetic field in combination with a cryogenically cooled probe enabled us to overcome the lack of cross peaks observed in nuclear Ö verhauser enhancement spectroscopy (NOESY) spectra acquired on less sensitive, lowerfield instruments 38 . The frequencies of all 1 H atoms (except those absent due to fast exchange with solvent) were assigned using homonuclear total correlation spectroscopy and NOESY spectra, while all protonated heteroatoms were assigned using heteronuclear HSQC spectra.…”

Differential Binding of Desmopressin and Vasopressin to Neurophysin-II

“…This is analogous to the amide proton of Phe 3 in our bound structure, which is directed to the inside of the 20-membered ring moiety. Similar observations were noted with the bound structure of oxytocin to neurophysin-I (6).…”

The INPHARMA technique for pharmacophore mapping: A theoretical guide to the method

“…For ligands that bind weakly to the target (K d in the lM range) detailed structural information on the ligand bound conformation can be obtained by transferred-NOEs and transferred-CCR rates [1][2][3][4][5][6]. This approach requires small quantities of unlabeled target (1-10 lM solution) and an excess of ligand, is applicable to any complex independently of the size of the target macromolecule and does not require any isotope labeling scheme.…”