SHP2是一种磷酸酶/适配蛋白，在各种信号通路中发挥着重要作用。 它的突变与癌症和发育性疾病有关。 SHP2包含一个蛋白酪氨酸磷酸酶（PTP）和两个SH2结构域。由于蛋白质组中具有多种同源蛋白质，选择性抑制这些结构域一直是具有挑战性的。 在这里，我们开发了一种单抗，合成结合蛋白，它结合并抑制了SHP2的PTP结构域。 它对SHP2 PTP具有选择性而非其同源蛋白。 单抗-PTP复合物的晶体结构表明，单抗同时结合了活性位点中高度保守的残基和周围不太保守的残基，从而解释了其高选择性。 它的表位与PTP和N端SH2结构域之间的界面重叠，在被自我抑制的SHP2中形成。 通过使用单抗作为PTP活性位点可及性的探针，我们开发了一种简单的非酶促测定SHP2的反调控。该测定表明，在没有激活的磷酸酪氨酸配体存在时，野生型SHP2和“PTP-dead”C459E突变体主要处于PPT活性位点难以访问的闭合状态，而E76K和C459S突变体处于开放活性状态。 它还揭示了先前开发的针对SH2结构域的单抗，缺乏磷酸酪氨酸配体，弱化了开放状态。这些结果为SHR2的构象平衡提供了证明，为表征和控制SHP2功能提供了有力工具，并为针对SHP2的药物发现提供了信息。 版权所有©2023作者。 由Elsevier Ltd.发表。 保留所有权利。

SHP2 is a phosphatase/adaptor protein that plays an important role in various signaling pathways. Its mutations are associated with cancers and developmental diseases. SHP2 contains a protein tyrosine phosphatase (PTP) and two SH2 domains. Selective inhibition of these domains has been challenging due to the multitude of homologous proteins in the proteome. Here, we developed a monobody, synthetic binding protein, that bound to and inhibited the SHP2 PTP domain. It was selective to SHP2 PTP over close homologs. A crystal structure of the monobody-PTP complex revealed that the monobody bound both highly conserved residues in the active site and less conserved residues in the periphery, rationalizing its high selectivity. Its epitope overlapped with the interface between the PTP and N-terminal SH2 domains that is formed in auto-inhibited SHP2. By using the monobody as a probe for the accessibility of the PTP active site, we developed a simple, nonenzymatic assay for the allosteric regulation of SHP2. The assay showed that, in the absence of an activating phospho-Tyr ligand, wild-type SHP2 and the "PTP-dead" C459E mutant were predominantly in the closed state in which the PTP active site is inaccessible, whereas the E76K and C459S mutants were in the open, active state. It also revealed that previously developed monobodies to the SH2 domains, ligands lacking a phospho-Tyr, weakly favored the open state. These results provide corroboration for a conformational equilibrium underlying allosteric regulation of SHP2, provide powerful tools for characterizing and controlling SHP2 functions, and inform drug discovery against SHP2.Copyright © 2023 The Author(s). Published by Elsevier Ltd.. All rights reserved.