酪氨酸磷酸酶Src同源性-2结构域含有蛋白酪氨酸磷酸酶-2(SHP2)的突变与多种人类疾病有关。SHP2中的大多数突变通过破坏其磷酸酶结构域和N末端SH2(磷酸酪氨酸识别)结构域之间的自抑制相互作用来增加其基础催化活性。相比之下,位于N端或C端SH2结构域的配体结合口袋中的一些疾病相关突变不会增加基础活性,并可能通过替代机制发挥其致病性.我们缺乏对这些SH2突变如何影响SHP2结构的分子理解,活动,和信号。这里,我们通过高通量生化筛选的组合表征了五个SHP2SH2结构域配体结合口袋突变体,生物物理和生化测量,和分子动力学模拟。我们表明,虽然这些突变中的一些改变了与磷酸化位点的结合亲和力,在N-SH2结构域中的T42A突变是独特的,因为它也实质上改变了配体结合特异性,尽管与SH2域的特异性决定区域相距8至10。该突变通过重塑磷酸酪氨酸结合口袋发挥其对序列特异性的影响,改变配体上磷酸酪氨酸和周围残基的接合模式。这种改变的特异性的功能结果是T42A突变体对激活配体的子集具有偏倚的敏感性并增强下游信号传导。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 Src homology-2 domain-containing protein tyrosine phosphatase-2 (SHP2) are associated with a variety of human diseases. Most mutations in SHP2 increase its basal catalytic activity by disrupting autoinhibitory 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 to 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.