Interaction of oxytocin with Ca2+: II. Proton magnetic resonance and molecular modeling studies of conformations of the hormone and its Ca2+ complex

Drastic changes in the CD and fluorescence spectra of oxytocin [cyclo(Cys1-Tyr2-Ile3-Gln4-Asn5-Cys6)-Pro7-Leu8-Gly 9-NH2] occur on binding Ca2+ in trifluoroethanol (Ananthanarayanan and Brimble, preceding paper). To further characterize the conformation of the Ca(2+)-bound hormone, we carried out 1H-nmr measurements in deuterated trifluorethanol of oxytocin and its 1:1 Ca2+ complex. The one-dimensional nmr data identified residues involved in Ca2+ binding and the extent of their perturbation on Ca2+ addition. The 3JNH-CH coupling constants and two-dimensional nuclear Overhauser effect (NOE) spectral cross peaks confirmed the helical nature of the Ca2+ complex deduced from CD data. Interproton distances in the free hormone and its Ca2+ complex were estimated from the respective NOE data. Apparent global minimum-energy conformations of free and Ca2+ bound oxytocin were computed using the Monte Carlo with energy minimization protocol, with and without incorporating the NOE-derived distance constraints. Taken together, our results show Ca2+ binding to oxytocin to be a two-step process. The binding of the first Ca2+ brings the otherwise extended tail segment of oxytocin closer to the ring moiety so that it wraps around the cation. This causes the maximal extent of change in all the spectral parameters. The subsequent formation of the 2:1 Ca-oxytocin complex results in the tail detaching itself away from the ring so as to bind the second Ca2+ ion. This leads to further spectral changes in the hormone molecule. The tail segment plays a major role in both steps. These observations may be useful in understanding the structural basis of oxytocin action.