INVOLVEMENT OF A POLYPROLINE HELIX-LIKE STRUCTURE IN THE INTERACTION OF 7B2 WITH PROHORMONE CONVERTASE-2

The neuroendocrine protein 7B2 is known to be involved in the biosynth esis and activity of prohormone convertase 2 (PC2). Previous studies h ave demonstrated that while the carboxyl-terminal portion of 7B2 (resi dues 155-186) regulates the enzymatic activity of PC2, the amino termi nus of the molecule (residues 1-151) is required for maturation of pro PC2. In this study we employed four different experimental approaches (co-immunoprecipitation with proPC2, facilitation of proPC2 maturation , acquisition of enzymatic activity, and thermal protection assays) to identify structural elements of 7B2 important for bioactivity. Inspec tion of the sequence of 7B2 indicated potential involvement of a polyp roline helix-like (PPII) structure, with similarities to those present within SH3 domain ligands, in the interaction of 7B2 with proPC2. Sit e-directed point mutagenesis of this proline-rich region confirmed the involvement of this area. Replacement of prolines in positions critic al to helix formation (Pro(90), Pro(91), Pro(93), and Pro(95)) either severely impaired or totally abolished 7B2 bioactivity, as gauged by t he four assays described. In addition, constructs longer than residues 1-121 were still functional, whereas those shorter than residues 1-10 9 were not. Computer-assisted analysis predicts the presence of an alp ha-helix structure between residues 107 and 123. We conclude that both the proline-rich region and the alpha-helix contribute to 7B2 activit y. Polyproline-containing peptides have been shown to be involved in c ytoplasmic protein-protein interactions; our results suggest that the polyproline helix motif may also be used to mediate protein-protein in teractions within the secretory pathway.