4-Fluoroproline derivative peptides: effect on PPII conformation and SH3 affinity

Area of Science:

• Biochemistry
• Molecular Biology
• Structural Biology

Background:

• Eukaryotic signal transduction relies on transient protein-protein complexes formed via modular domains.
• Pro-rich sequences bind SH3 domains by adopting a polyproline II (PPII) helix conformation.
• Short Pro-rich peptides often lack pre-formed secondary structure, and binding incurs entropic costs.

Purpose of the Study:

• To stabilize the PPII helix conformation of the Pro-rich HPK1 decapeptide (P2) using fluorinated proline analogues.
• To investigate the impact of these modifications on peptide-protein interactions with the HS1 SH3 domain.
• To understand how proline modifications influence binding affinity and PPII helix propensity.

Main Methods:

• Synthesis of P2 analogues with 4(S)- or 4(R)-4-fluoro-L-proline at specific proline positions.
• Quantitative analysis of peptide-SH3 domain interactions using the NMR-based titration with ligand-induced chemical shift analysis (NILIA) and Circular Dichroism (CD) spectroscopy.
• CD thermal analysis to assess the propensity of analogues to adopt the PPII helix conformation.

Main Results:

• Contrary to expectations, analogues exhibited lower K(d) values (higher affinity) than the parent peptide P2.
• The induction of a stable PPII helix conformation was insufficient to enhance binding affinity.
• The effect of 4-fluoroproline on binding affinity was highly dependent on its position within the peptide sequence and its stereochemistry (chirality).

Conclusions:

• Stabilizing the PPII helix conformation of short Pro-rich peptides does not automatically increase their affinity for SH3 domains.
• The precise positioning and stereochemistry of modifications like 4-fluoroproline are critical determinants of binding affinity.
• These findings provide insights into the nuanced structural requirements for Pro-rich peptide recognition by SH3 domains.