Ionizable networks mediate pH-dependent allostery in the SH2 domain–containing signaling proteins SHP2 and SRC

Intracellular pH dynamics regulate many cell biological processes. We developed a computational pipeline to identify pH-sensitive proteins and the molecular mechanisms that regulate their pH-dependent activity. By applying the pipeline to the phosphatase SHP2, which regulates signaling pathways that control pH-dependent cellular processes, we found that SHP2 phosphatase activity was sensitive to pH in vitro and in cells and that mutation of His¹¹⁶ and Glu²⁵² abolished SHP2 pH-sensitive activity. We also found that the activity of the kinase SRC was pH dependent and that mutations in a network of ionizable amino acids abolished pH-sensitive activity. Furthermore, we found that SRC kinase activity was pH sensitive even in the presence of the growth factor EGF, which stimulates SRC activity in a phosphorylation-dependent manner, or with a phosphomimetic substitution (Y527E) that promotes SRC autoinhibition. These data suggest that pH-sensitive regulation functions in concert with established phosphorylation-dependent mechanisms to regulate SRC kinase activity. Constant pH molecular dynamics simulations performed on both SHP2 and SRC supported allosteric regulation mediated by pH-dependent binding of inhibitory SH2 domains to the respective catalytic domain in each protein. We also identified evolutionarily conserved putative pH-sensing networks in other SH2 domain-containing signaling proteins. Together, our computational, biophysical, and cellular analyses reveal a role for intracellular pH dynamics in allosterically regulating the activities of modular SH2 signaling proteins to control cell biology.