Why the first self-binding peptide of human c-Src kinase does not contain class II motif but can bind to its cognate Src homology 3 domain in class II mode?

Src homology 3 (SH3) domains are small protein modules involved in the regulation of important cellular pathways such as proliferation and migration, which canonically prefer to recognize and interact with proline-rich peptide ligands with class I or class II motif. Previously, we identified two self-binding peptides (SBPs) in human c-Src tyrosine kinase, of which the first SBP (fSBP) segment ((248)SKPQTQGLAK(257)) fulfills intramolecular interaction with the kinase SH3 domain to regulate the kinase function. The segment (and its equivalents in other c-Src family members) does not contain canonical class II motif (PxxQxL versus PxxPx+), but can bind to SH3 domain in a routine class II mode. Existing theories such as non-polyproline-II binding conformation, unusual peptide-binding pocket and extensive use of contacts cannot explain this atypical recognition phenomenon. Here, we performed a systematic investigation of SH3-fSBP binding in different conditions, including the segment in full-length kinase or in isolated state, the kinase in different forms and the fSBP residue mutations, by using microsecond molecular dynamics simulations, conformational clustering analyses and binding energetics calculations. We purposed a new mechanism that the protein context is primarily responsible for the atypical intramolecular SH3-fSBP recognition in c-Src kinase, which can promote the tight packing of segment against domain surface, support the segment polyproline-II (PPII) conformation in unbound state, and avoid unfavorable segment interactions with SH3 charged region by forming a C-terminal t-turn. In addition, the only proline residue Pro250 of fSBP segment is also required for the segment recognition by SH3 domain in c-Src kinase context; lack of Pro250 residue the segment exhibits considerable disorder and cannot maintain in PPII helical conformation, thus largely impairing the domain-segment binding capability. Further binding analysis confirms that the isolated fSBP peptide cannot bind effectively to SH3 domain out of kinase context, whereas its mutant version, i.e. fSBP(Q253P/L255R) peptide, which possesses the canonical class II motif, exhibits an increased affinity to the domain. Communicated by Ramaswamy H. Sarma

Automated summary

The mechanism of SH3-fSBP binding primarily depends on protein context, promoting tight packing and avoiding unfavorable interactions between the segment and SH3 domain through the formation of a C-terminal t-turn.