Dynamics simulation, energetics calculation and experimental analysis of the intermolecular interaction between human neonatal ABL SH3 domain and its N -substituted peptoid ligands.

Non-receptor tyrosine kinase of neonatal ABL (nABL) is distributed in the nucleus and cytoplasm of proliferating cells in embryo and neonate, and has been implicated in the pathogenesis of neonatal leukemia and other hematological diseases. The kinase contains a regulatory Src homology 3 (SH3) domain that can specifically recognize proline-rich peptide segments on its partner protein surface. In this study, we systematically investigated the N -substitution effect on the binding of an empirically designed proline-rich peptide p9 to nABL SH3 domain by integrating dynamics simulations, energetics calculations and fluorescence affinity assays. The p9 is an almost all proline-composed decapeptide, with only a sole tyrosine at its residue 4, which has been found to bind nABL SH3 domain at a micromolar level in a class I mode. Here, the non-key residues of p9 peptide were independently replaced by various N -substituted amino acids to create a systematic N -substitution profile, from which we can identify those favorable, neutral and unfavorable substitutions at each peptide residue. On this basis a combinatorial peptoid library was rationally designed by systematically combining the favorable N -substituted amino acids at non-key residues of p9 peptide, thus resulting in a number of its peptoid counterparts. The binding affinity of top peptoid hits was observed to be comparable with or improved moderately relative to p9 peptide, with K _d ranging between 3.1 and 76 μM. Structural analysis revealed that the peptoids can be divided into exposed, polar and hydrophobic regions from N- to C-termini, in which the polar and hydrophobic regions confer specificity and stability to the domain-peptoid interaction, respectively. In addition, a designed peptoid was also observed to exhibit 5.3-fold SH3-selectivity for nABL over cSRC, suggesting that the N -substitution can be used to improve not only binding affinity but also recognition specificity of SH3 binders.Communicated by Ramaswamy H. Sarma.