Effect of 4-R-hydroxy-L-proline (Hyp) on peptide conformation and SH3 affinity

Short proline-rich peptides interact with SH3 domains, exhibiting little or no secondary structure before their binding to the cognate proteintargets. Under these conditions the binding process of a proline-rich peptide with the SH3 domain shows unfavorable binding entropy, likely resulting from a loss of rotational freedom on the formation of the PPII helix. With the aim of stabilizing the PPII helix conformation in SH3 binding motifs, in the previous years, we replaced the proline residues of the HPK1 proline-rich decapeptide, PPPLPPKPKF (P2), either with 4-R- (Fp) or with 4-S- (fp) fl uoro-L-proline at different i, i+3 positions. The interactions of the fluoroproline-peptides with the SH3 domain of cortactin protein were analyzed quantitatively by Non-Immobilized Ligand Interactions Assay by Circular Dichroism (NILIA-CD), whereas CD thermal transitions were measured to correlate their propensity to adopt PPII helix with their affi nity for SH3. Results show that although the introduction of the Fp residue stabilizes the PPII helix conformation of peptides in a position-dependent manner, the induction of a stable peptide conformation does not increase the ligand affinity towards the SH3 domain of cortactin. To explore the effect of electron-withdrawing substituent in the Pro residue, the Fp residues of P2 were replaced by the natural amino acid 4-R-hydroxy-proline (Hyp). Unexpectedly, NILIACD and CD thermal transitions results showed that the Hyp-containing peptides exhibit a stable conformation in aqueous buffer and Kd values lower than the corresponding Fp peptide-analogues. In particular, Hyp3 peptide containing Hyp residues at i, i+3 and i+6 positions adopts the greater percentage of PPII helix conformation over the entire studied temperature range and shows a Kd value comparable to that of the parent P2 peptide.