Padua, R. | bioRxiv Science

bioRxiv2026

Graphene Device for High-Temperature Crystallography Visualizes How Enzymatic Reactivity is Rooted in Ensemble Probability

Life exists at temperatures ranging from -20 to 122 {degrees}C. However, the majority of high-resolution structural data in the Protein Data Bank (PDB) were obtained at cryogenic temperatures, where biological function is halted due to the lack of thermal fluctuations. To overcome this fundamental problem and directly link structure to biological function, we have created a graphene-based device that significantly extends the temperature range for high-resolution macromolecular X-ray diffraction data collection. Using the new device, we obtained models of the transition state ensembles for a psychrophilic, a mesophilic, and a thermophilic homolog of the enzyme orotidine 5-monophosphate decarboxylase from -173 to 65 {degrees}C. The data reveal how the active site ensemble structure at the transition state of each homolog changes with temperature, directly visualizing how the measured catalytic rates are rooted in the ensemble probabilities of reactive distances. The multi-temperature transition state ensembles further illuminate why cryogenic data, although useful, are inaccurate for describing biological processes.

biophysics↗

bioRxiv2025

Phosphatase SHP2 pathogenic mutations enhance activity by altering conformational sampling

SH2 domains are critical mediators of cellular signaling, although the molecular mechanisms by which they bind their phosphopeptide ligands remain incompletely understood. We investigate the atomic mechanisms underlying both healthy regulation and dysregulation of the human protein tyrosine phosphatase SHP2, a key regulator of cellular signaling. While most pathogenic mutations cluster near the PTP/N-SH2 interface, the E139D and T42A mutations are located within the regulatory SH2 domains, and their mechanisms of dysregulation remain controversial. The T42A mutation in the N-SH2 domain paradoxically increases phosphotyrosine-peptide binding affinity despite disrupting the hydrogen bond of T42 to the phosphoryl group, a puzzling contradiction that remains unresolved. We find that the T42A mutation shifts the conformational ensemble of peptide-bound N-SH2 toward a zipped {beta}-sheet state and suppresses millisecond conformational exchange, supporting a model in which enhanced stabilization of the zipped conformation contributes to hyperactivation. This conformational shift provides a structural rationale for the increased affinity of T42A and helps reconcile previously conflicting models of peptide-induced SHP2 activation. By integrating X-ray ensemble refinement with NMR relaxation, our work illustrates how complementary structural and dynamic approaches can uncover regulatory mechanisms in SHP2 and may inform broader principles of SH2-mediated phosphopeptide recognition. Significance StatementHere, we characterize how two pathogenic SH2-domain mutations alter SHP2 regulation and lead to hyperactivation. We identify a previously unobserved apo conformation of the N-SH2 domain in which Tyr66 occludes the peptide-binding cleft, indicating that a conformational change is required for full binding of activating phosphopeptides. Our data suggest that the T42A mutation shifts the equilibrium toward a zipped central {beta}-sheet state in the peptide-bound N-SH2 domain as the most likely model underlying the measured 10-fold increased binding affinity. These results help clarify the structural basis for SHP2 regulation and illustrate how conformational dynamics shape SH2-phosphopeptide recognition.

biophysics↗