PDF(Pubmed)
Abstract：
Multi-domain enzymes can be regulated by both inter-domain interactions and structural features intrinsic to the catalytic domain. The tyrosine phosphatase SHP2 is a quintessential example of a multi-domain protein that is regulated by inter-domain interactions. This enzyme has a protein tyrosine phosphatase (PTP) domain and two phosphotyrosine-recognition domains (N-SH2 and C-SH2) that regulate phosphatase activity through autoinhibitory interactions. SHP2 is canonically activated by phosphoprotein binding to the SH2 domains, which causes large inter-domain rearrangements, but autoinhibition can also be disrupted by disease-associated mutations. Many details of the SHP2 activation mechanism are still unclear, the physiologically-relevant active conformations remain elusive, and hundreds of human variants of SHP2 have not been functionally characterized. Here, we perform deep mutational scanning on both full-length SHP2 and its isolated PTP domain to examine mutational effects on inter-domain regulation and catalytic activity. Our experiments provide a comprehensive map of SHP2 mutational sensitivity, both in the presence and absence of inter-domain regulation. Coupled with molecular dynamics simulations, our investigation reveals novel structural features that govern the stability of the autoinhibited and active states of SHP2. Our analysis also identifies key residues beyond the SHP2 active site that control PTP domain dynamics and intrinsic catalytic activity. This work expands our understanding of SHP2 regulation and provides new insights into SHP2 pathogenicity.
摘要：
多结构域酶可以通过结构域间相互作用和催化结构域固有的结构特征来调节。酪氨酸磷酸酶SHP2是由域间相互作用调节的多结构域蛋白的典型实例。该酶具有蛋白酪氨酸磷酸酶（PTP）结构域和两个磷酸酪氨酸识别结构域（N-SH2和C-SH2），它们通过自抑制性相互作用调节磷酸酶活性。SHP2通过与SH2结构域结合的磷蛋白被规范激活，这导致了大的域间重排，但自身抑制也可能被疾病相关突变破坏。SHP2激活机制的许多细节仍不清楚，生理相关的活性构象仍然难以捉摸，和数百种SHP2的人类变体尚未被功能表征。这里,我们对全长SHP2及其分离的PTP域进行深度突变扫描，以检查突变对域间调控和催化活性的影响。我们的实验提供了SHP2突变敏感性的全面图谱，在存在和不存在域间调控的情况下。再加上分子动力学模拟,我们的研究揭示了控制SHP2自抑制和活性状态稳定性的新结构特征。我们的分析还确定了SHP2活性位点之外的控制PTP结构域动力学和固有催化活性的关键残基。这项工作扩展了我们对SHP2调控的理解，并为SHP2致病性提供了新的见解。
