Polyproline II Helix Research Articles

Critical residues in an SH3 domain from Sem-5 suggest a mechanism for proline-rich peptide recognition.

Src homology 3 (SH3) domains bind specific proline-rich peptide motifs. To identify interactions involved in peptide recognition, we have mutated residues on the putative binding surface of an SH3 domain from the Caenorhabditis elegans protein Sem-5. Among the most critical positions are three adjacent aromatic residues, which appear to participate in highly stereospecific packing interactions with the ligand. The co-planar arrangement of two of these residues closely matches the periodicity of a poly-proline II (PPII) helix. Thus, a model for recognition has the peptide adopting a PPII helix, with the pyrrolidine rings on one helical face interlocking with the aromatic SH3 residues.

Interaction of prolyl 4-hydroxylase with synthetic peptide substrates. A conformational model for collagen proline hydroxylation.

With the aim of understanding the structural basis for the substrate specificity of collagen prolyl 4-hydroxylase, we have studied the conformational features of synthetic oligopeptide substrates and their interaction with the enzyme purified from chicken embryo. Circular dichroism and infrared spectral data, taken in conjunction with relevant crystal structure data, indicated an equilibrium mixture of the polyproline-II (PP-II) helix, the beta-turn, and the random coil conformations in aqueous and trifluoroethanol solutions of the "collagen-related" peptides: t-Boc-Pro-Pro-Gly-Pro-OH, t-Boc-Pro-Pro-Gly-Pro-NHCH3, t-Pro-Pro-Gly-Pro-Pro-OH, t-Boc-Pro-Pro-Ala-Pro-OH, and t-Boc-Pro-Pro-Gln-Pro-OCH3, where t-Boc is tert-butoxycarbonyl. In another set of peptides related to elastin, t-Boc-Val-Pro-Gly-Val-OH and t-Boc-Gly-Val-Pro-Gly-Val-OH, the data indicated the beta-structure, rather than the PP-II helix, was in equilibrium with the beta-turn. Kinetic parameters for the enzymatic hydroxylation of the peptides showed that as a group, the first (proline-rich) set of peptides has higher Km values and lower Vmax and Kcat/Km values than the valine-rich peptides. Data on the inhibition of hydroxylation of the standard assay substrate (Pro-Pro-Gly)10 by the oligopeptides pointed to common binding sites for the peptides. Hydroxyproline-containing peptides had no effect on the hydroxylation of the standard substrate, showing the absence of product inhibition. Based on these and earlier data, we propose that in collagen and related peptides, a supersecondary structure consisting of the PP-II helix followed by the beta-turn is the minimal structural requirement for proline hydroxylation. The PP-II structure would aid effective interaction at the substrate binding subsites, while the beta-turn would be essential at the catalytic site of the enzyme. In elastin and related peptides, the beta-strand structure may be interchangeable with the PP-II structure. This conformational model for proline hydroxylation resolves the discrepancies in earlier proposals on the substrate specificity of prolyl 4-hydroxylase. It is also consistent with the available information on the active site geometry of the enzyme.