Structural studies of a neuropeptide precursor protein with an RGD proteolytic site

The snail Lymnaea stagnalis produces a neuropeptide precursor protein that contains seven Arg-Gly-Asp (RGD) sites. These sites are recognized and clea ved by one or more prohormone convertases in the first processing step to y ield mature neuropeptides in the secretory pathway. Conformations of two sy nthetic RGD-containing peptides derived from the L. stagnalis precursor pro tein were determined by NMR spectroscopy. The peptides were tested in a pla telet aggregation assay for RGD activity and were processed in vitro by PC2 and furin. The native peptide with a proline following the RGD site has mi nimal structure around the RGD region, does not inhibit platelet aggregatio n, and is properly processed by the enzymes PC2 and furin. A variant of the native fragment with a serine following the RGD sequence has a significant amount of a reverse turn around the RGD region, is a potent inhibitor of p latelet aggregation, and is processed with the same specificity as the nati ve fragment. The large conformational differences between the two peptides provide a molecular mechanism for effects of proline residues following the RGD site and suggest that precursor processing is influenced more by flexi bility than by the conformation of the processing site.