The impact of either 4-R-hydroxyproline or 4-R-fluoroproline on the conformation and SH3m-cort binding of HPK1 proline-rich peptide.

SH3 domains are probably the most abundant molecular-recognition modules of the proteome. A common feature of these domains is their interaction with ligand proteins containing Pro-rich sequences. Crystal and NMR structures of SH3 domains complexes with Pro-rich peptides show that the peptide ligands are bound over a range of up to seven residues in a PPII helix conformation. Short proline-rich peptides usually adopt little or no ordered secondary structure before binding interactions, and consequently their association with the SH3 domain is characterized by unfavorable binding entropy due to a loss of rotational freedom on forming the PPII helix. With the aim to stabilize the PPII helix conformation into the proline-rich decapeptide PPPLPPKPKF (P2), we replaced some proline residues either with the 4(R)-4-fluoro-L-proline (FPro) or the 4(R)-4-hydroxy-L-proline (Hyp). The interactions of P2 analogues with the SH3 domain of cortactin (SH3(m-cort)) were analyzed by circular dichroism spectroscopy, while CD thermal transition experiments have been used to determine their propensity to adopt a PPII helix conformation. Results show that the introduction of three residues of Hyp efficiently stabilizes the PPII helix conformation, while it does not improve the affinity towards the SH3 domain, suggesting that additional forces, e.g., electrostatic interactions, are involved in the SH3(m-cort) substrate recognition.