
==== Front
bioRxiv
BIORXIV
bioRxiv
2692-8205
Cold Spring Harbor Laboratory

10.1101/2024.08.21.608875
preprint
1
Article
Ionizable networks mediate pH-dependent allostery in SH2 signaling proteins
Van Dyck Papa Kobina http://orcid.org/0000-0002-2998-9508

Piszkin Luke http://orcid.org/0000-0002-6997-1070

Gorski Elijah A.
Nascimento Eduarda Tartarella
Abebe Joshua A.
Hoffmann Logan M.
Peng Jeffrey W.
White Katharine A. http://orcid.org/0000-0002-5831-1886

21 8 2024
2024.08.21.608875https://creativecommons.org/licenses/by-nc-nd/4.0/ This work is licensed under a Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International License, which allows reusers to copy and distribute the material in any medium or format in unadapted form only, for noncommercial purposes only, and only so long as attribution is given to the creator.
http://biorxiv.org/lookup/doi/10.1101/2024.08.21.608875
nihpp-2024.08.21.608875.pdf
Introduction

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.
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