酪氨酸磷酸酶SHP2中的大多数疾病相关突变通过使其自抑制状态不稳定而增加其基础催化活性。相比之下,一些突变不会增加SHP2的基础活性,并可能通过替代机制发挥其致病性.我们缺乏对这些突变如何影响SHP2结构的分子理解,活动,和信号。这里,我们使用高通量生化筛选,表征了在SHP2的调节磷酸酪氨酸识别结构域的配体结合口袋内发生的五个这样的突变,生物物理和生化测量,分子动力学模拟,和细胞测定。虽然许多这些突变影响结合亲和力,T42A突变的独特之处在于它还改变了配体结合特异性。因此,T42A突变体对激活磷蛋白的一部分具有偏倚敏感性.我们的研究强调了疾病相关突变的细微差别作用机制的一个例子,其特征是蛋白质-蛋白质相互作用特异性的变化,从而改变酶的激活。
Mutations in the tyrosine phosphatase SHP2 are associated with a variety of human diseases. Most mutations in SHP2 increase its basal catalytic activity by disrupting auto-inhibitory interactions between its phosphatase domain and N-terminal SH2 (phosphotyrosine recognition) domain. By contrast, some disease-associated mutations located in the ligand-binding pockets of the N- or C-terminal SH2 domains do not increase basal activity and likely exert their pathogenicity through alternative mechanisms. We lack a molecular understanding of how these SH2 mutations impact SHP2 structure, activity, and signaling. Here, we characterize five SHP2 SH2 domain ligand-binding pocket mutants through a combination of high-throughput biochemical screens, biophysical and biochemical measurements, and molecular dynamics simulations. We show that, while some of these mutations alter binding affinity to phosphorylation sites, the T42A mutation in the N-SH2 domain is unique in that it also substantially alters ligand-binding specificity, despite being 8-10 Å from the specificity-determining region of the SH2 domain. This mutation exerts its effect on sequence specificity by remodeling the phosphotyrosine binding pocket, altering the mode of engagement of both the phosphotyrosine and surrounding residues on the ligand. The functional consequence of this altered specificity is that the T42A mutant has biased sensitivity toward a subset of activating ligands and enhances downstream signaling. Our study highlights an example of a nuanced mechanism of action for a disease-associated mutation, characterized by a change in protein-protein interaction specificity that alters enzyme activation.