Distal Mutations Rewire Allosteric Networks to Control Substrate Specificity in PTP1B

), in some cases reversing substrate preference relative to the wild-type enzyme. Solution NMR spectroscopy and microsecond molecular dynamics simulations demonstrate that these mutations perturb long-range communication networks, disrupting coupling between helices α3 and α7 and altering acid-loop flexibility and active-site dynamics. Notably, the E297A mutation has the most pronounced effects, rigidifying the acid loop and weakening allosteric communication to the catalytic center. Community network analysis highlights the acid loop and helix α7 as central hubs linking distal sites to the active site. Together, these results establish that distal mutations can reshape PTP1B's dynamic landscape, thereby modulating substrate specificity. This work expands our understanding of allosteric regulation in PTP1B and provides a framework for targeting dynamic networks to control phosphatase activity.