Charactering ionizable residue networks at SH2 interfaces in pH-sensitive proteins

Intracellular pH (pHi) dynamics (7.0 to 7.8) regulate myriad cell processes but the molecular mechanisms driving these behaviors are largely unknown. While some pH-sensitive proteins use titratable histidine residues to sense physiological changes in pHi, others have unknown or histidine-independent mechanisms. Charged amino acids (Asp, Glu, and Lys) can have pKas that are up- or down-shifted into the physiological range depending on protein environment, but the role for these residues in regulating pH-dependent protein function is understudied. Here, we use computational and biochemical approaches to investigate pH sensors that use networks of cooperative ionizable residues with a cumulative physiological pKa. We performed computational analyses of two pH sensitive signaling proteins with unknown mechanisms: (phosphatidylinositol-3-kinase (PI3K) and SHP-2 tyrosine-protein phosphatase). In both proteins, we identified networks of ionizable residues with upshifted pKas at the inhibitory SH2 domain interface. We next used biochemical mutational analyses to probe the accuracy of our computational approaches by measuring pH sensitive activity of wild-type and mutant proteins in vitro. Importantly, because SH2 domains are structurally conserved, this pH-sensing mechanism could be conserved in a subset of kinases and phosphatases with inhibitory SH2 domains. We tested this hypothesis by identifying a network of ionizable residues in other signaling proteins that contain inhibitory SH2 domains. We analyzed JAK2 kinase, which has not been shown to have pH sensitive function, and identified an ionizable network at the SH2 domain interface. Future work will validate this JACK2 result biochemically and explore whether our computational approaches can be applied to identify pH sensitive proteins across the human proteome. Improved understanding of how ionizable networks respond to pH could reveal paths to better therapeutic targeting of these proteins in diseases with dysregulated pHi.