Structural And Energetic Insight Into Proline-Mediated Conformational Constraint In Protein-Peptide Recognition

Given the fact that protein–protein interactions(PPIs)generally have a flat,large and hydrophobic interface that is lack of pocket and groove to accommodate small-molecule ligands,chemical and protein drugs are difficult to mediate the PPIs by binding their complex interface.Instead,peptide-like agents seem to be more suitable and as “natural” candidates to act as the potential modulators of PPIs.However,isolated polypeptide molecules tend to possess large flexibility and intrinsic disorder,which require additional conformational constraints to interact effectively with their protein targets in a stable and specific manner.Previously,the chemical strategies such as cyclization and hydrocarbon stapling have been widely used to constrain peptide conformation and to reduce peptide flexibility in isolated state.These methods,however,are synthetically complicated.And we should introduce natural chemical moieties to the peptides.In the present study,we have systematically investigated the molecular role of proline in peptide conformational flexibility and in protein–peptide recognition at structural,energetic and dynamic levels.Proline is a natural amino acid that can prevent the rotation of backbone C–N bond by its cyclized side-chain,thus largely minimizing the backbone flexibility.Proline-rich peptides(PRP)can spontaneously fold into a flexibility-reduced polyproline Ⅱ(PPⅡ)helical conformation.Here,a variety of PRP and their interaction with protein targets are modeled,analyzed and characterized using long-term molecular dynamics(MD)simulations and binding free energy decomposition.We systematically explore the structural and energetic effects of the abundance,pattern and arrangement of proline residues on peptide flexibility and protein–peptide interaction.It is revealed that the proline should play a crucial role in medicating the stability and specificity of protein–peptide recognition,but the underlying mechanism is very complicated.The peptide proline residues usually do not directly contact protein target,which,instead,influence the protein–peptide interaction through indirectly manner such as pre-organization of peptide conformation before protein–peptide binding and minimization of entropy penalty upon the binding.It is also found from MD simulations that there is an inclination of spontaneous dissociation between protein and peptide when the prolines are virtually mutated to other residues.Furthermore,we chose the proto-oncogene non-receptor tyrosine kinase c-Src as a case study to perform structure-based rational design of potent c-Src SH3-binding PRP peptides.It is revealed that the protein context plays an important role in the intramolecular interaction between the c-Src SH3 domain and its self-binding peptide(SBP);the context can constrain the peptide into PPⅡ conformation and thus facilitate its binding to SH3 domain.Stripping the SBP from protein context would substantially impair SH3 affinity by increasing entropy penalty upon the domain–peptide binding.The SBP is identified to bind SH3 domain in a class Ⅱ manner,and on this basis,we derived a series of modified versions of the wild-type peptide.These modified peptide mutants have been structurally optimized with respect to their molecular flexibility and interaction potency with SH3 domain,in order to minimize indirect entropy penalty and to maximize direct binding enthalpy simultaneously.Consequently,several rationally designed peptides were obtained,which exhibit a moderately or considerably increased affinity relative to the wild-type peptide.