Evidence for the presence of a secondary structure at the dibasic processing site of prohormone: The pro-ocytocin model

Bioactivation of pro-proteins by limited proteolysis is a general mechanism in the biosynthesis of hormones, receptors and viral protein precursors. This proceeds by cleavage of peptide bonds at the level of single or pairs of basic residues in the preforms. Examination of a number of cleavage loci in various precursors failed to reveal any consensus primary sequence around the dibasic cleavage sites. Thus it has been proposed, on the basis of secondary structure predictions [Rholam,M., Nicolas,P. and Cohen,P. (1986) FEBS Lett., 207, 1-6], that those basic residues which operate as signal loci for the proteolytic enzyme machinery are situated in, or next to, privileged precursor regions most often constituted by flexible and exposed motifs, e.g. β-turns and/or loops. Peptides reproducing the N-terminal processing domain of the hormone precursor, pro-ocytocin-neurophysin, were examined by a combination of spectroscopical techniques including circular dichroism, infrared Fourier transform and one- and two-dimensional proton NMR. The results indicate that: (i) the region situated on the N terminus of the Lys-Arg doublet is organized as a β-turn in solution; (ii) the sequential organization of the residues participating in the β-turn determines the privileged relative orientation of the basic amino acid side chains and the subtype of turn; (iii) the peptide segment situated on the C-terminal side of the dibasic, corresponding to the N-terminal octapeptide of neurophysin, is organized as an α-helix. It is concluded that the β-turn situated on the N terminus of the dibasic cleavage site is an important feature for processing enzyme recognition and that the respective structures situated on both sides of the β-turn determine the degree of exposure and accessibility of the processing site. Furthermore the data suggest that the subtype of turn and the relative orientation of the Lys and Arg side chains do not play a direct role in substrate recognition.