Ionizable networks mediate pH-dependent allostery in SH2 signaling proteins.

Transient intracellular pH dynamics 1 regulate mammalian proliferation 2,3 , migration 4 , and differentiation 5 . However, for many pH-dependent cell processes, the molecular mediators are unknown 6 . Prior work identified histidine residues as molecular switches in pH-sensitive proteins, but how other ionizable residues contribute to pH-dependent protein allostery is understudied. Here, we develop an in silico computational pipeline to identify putative pH-sensitive proteins and their molecular mechanisms. We first apply this pipeline to SHP2, a known pH-sensitive signaling protein with an uncharacterized molecular mechanism. We show wild-type SHP2 phosphatase activity is pH-sensitive in vitro and in cells, and mutation of identified H116 and E252 to non-titratable alanine residues abolishes pH-sensitive function. We also show that c-Src is a previously unrecognized pH-dependent kinase, and mutation of the identified ionizable network again abolishes pH-sensitive activity. Constant pH molecular dynamics simulations support a conserved allosteric mechanism of pH-dependent binding of inhibitory SH2 domains to the functional catalytic domains of SHP2 and c-Src. We apply our computational pipeline across SH2 domain-containing signaling proteins and identify evolutionarily conserved putative pH-sensing networks. Our results reveal that pH is an allosteric regulator of SH2 domain-containing signaling proteins providing insight into normal pH-dependent cell biology and diseases where pHi is dysregulated, such as cancer.