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Abstract
Protein tyrosine phosphatase SHP2 functions as a key regulator of cell cycle control, and activating mutations cause several cancers. Here, we dissect the energy landscape of wild-type SHP2 and the oncogenic mutation E76K. NMR spectroscopy and X-ray crystallography reveal that wild-type SHP2 exchanges between closed, inactive and open, active conformations. E76K mutation shifts this equilibrium toward the open state. The previously unknown open conformation is characterized, including the active-site WPD loop in the inward and outward conformations. Binding of the allosteric inhibitor SHP099 to E76K mutant, despite much weaker, results in an identical structure as the wild-type complex. A conformational selection to the closed state reduces drug affinity which, combined with E76K’s much higher activity, demands significantly greater SHP099 concentrations to restore wild-type activity levels. The differences in structural ensembles and drug-binding kinetics of cancer-associated SHP2 forms may stimulate innovative ideas for developing more potent inhibitors for activated SHP2 mutants.
Highlights
Protein tyrosine phosphatase SHP2 functions as a key regulator of cell cycle control, and activating mutations cause several cancers
We utilized a combination of nuclear magnetic resonance (NMR) spectroscopy, X-ray crystallography, smallangle X-ray scattering (SAXS), enzyme kinetics, isothermal titration calorimetry (ITC), and stopped-flow kinetics to show that SHP2 exists in a dynamic equilibrium between a closed state and an open state
We describe two structural features for SHP2: (i) the structure of the open, active conformation of SHP2 with a protein tyrosine phosphatase domain (PTP)/ C-SH2 interface that is vastly different from the interface of the inactive state, and the N-SH2 detached from PTP; and (ii) direct detection of the inward conformation of the active-site WPD loop (WPD-in) in the ligand-free protein that was previously seen only in an outward conformation (WPD-out) in SHP2
Summary
Protein tyrosine phosphatase SHP2 functions as a key regulator of cell cycle control, and activating mutations cause several cancers. NMR spectroscopy and X-ray crystallography reveal that wild-type SHP2 exchanges between closed, inactive and open, active conformations. A conformational selection to the closed state reduces drug affinity which, combined with E76K’s much higher activity, demands significantly greater SHP099 concentrations to restore wild-type activity levels. An allosteric inhibitor (SHP099) was developed for the nonreceptor protein tyrosine phosphatase SHP21,2, a fundamental enzyme for cell cycle control, and the root of many pathologies such as LEOPARD syndrome, Noonan syndrome (NS)[3,4,5], and juvenile myelomonocytic leukemia[6,7]. We further show that the SHP099 inhibitor binds via a pure conformational selection mechanism, associating only with the closed state, and, the oncogenic mutations vastly reduce the inhibitor affinity
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