Institute of Structural and Molecular Biology, Division of Biosciences, University College London, London, United Kingdom; The Francis Crick Institute, London, United Kingdom.
Institute of Structural and Molecular Biology, Division of Biosciences, University College London, London, United Kingdom.
Biophys J. 2018 Jul 3;115(1):31-45. doi: 10.1016/j.bpj.2018.05.031.
Phosphatidylinositol phospholipase Cγ (PLCγ) is an intracellular membrane-associated second-messenger signaling protein activated by tyrosine kinases such as fibroblast growth factor receptor 1. PLCγ contains the regulatory γ-specific array (γSA) comprising a tandem Src homology 2 (SH2) pair, an SH3 domain, and a split pleckstrin homology domain. Binding of an activated growth factor receptor to γSA leads to Tyr783 phosphorylation and consequent PLCγ activation. Several disease-relevant mutations in γSA have been identified; all lead to elevated phospholipase activity. In this work, we describe an allosteric mechanism that connects the Tyr783 phosphorylation site to the nSH2-cSH2 junction and involves dynamic interactions between the cSH2-SH3 linker and cSH2. Molecular dynamics simulations of the tandem SH2 protein suggest that Tyr783 phosphorylation is communicated to the nSH2-cSH2 junction by modulating cSH2 binding to sections of the cSH2-SH3 linker. NMR chemical shift perturbation analyses for designed tandem SH2 constructs reveal combined fast and slow dynamic processes that can be attributed to allosteric communication involving these regions of the protein, establishing an example in which complex N-site exchange can be directly inferred from H,N-HSQC spectra. Furthermore, in tandem SH2 and γSA constructs, molecular dynamics and NMR results show that the Arg687Trp mutant in PLCγ1 (equivalent to the cancer mutation Arg665Trp in PLCγ2) perturbs the dynamic allosteric pathway. This combined experimental and computational study reveals a rare example of multistate kinetics involved in a dynamic allosteric process that is modulated in the context of a disease-relevant mutation. The allosteric influences and the weakened binding of the cSH2-SH3 linker to cSH2 should be taken into account in any more holistic investigation of PLCγ regulation.
磷酸肌醇磷脂酶 Cγ（PLCγ）是一种细胞内膜相关的第二信使信号蛋白，可被成纤维细胞生长因子受体 1 等酪氨酸激酶激活。PLCγ包含由串联 Src 同源 2（SH2）对、SH3 结构域和分裂的pleckstrin 同源结构域组成的调节γ特异性阵列（γSA）。激活的生长因子受体与γSA 的结合导致 Tyr783 磷酸化，进而导致 PLCγ 的激活。已经鉴定出 γSA 中的几种与疾病相关的突变；所有这些突变都导致磷脂酶活性升高。在这项工作中，我们描述了一种连接 Tyr783 磷酸化位点和 nSH2-cSH2 连接点的变构机制，涉及 cSH2-SH3 接头和 cSH2 之间的动态相互作用。串联 SH2 蛋白的分子动力学模拟表明，Tyr783 磷酸化通过调节 cSH2 与 cSH2-SH3 接头的部分结合，将信息传递到 nSH2-cSH2 连接点。针对设计的串联 SH2 结构的 NMR 化学位移扰动分析揭示了快速和缓慢的动态过程，这些过程可以归因于涉及该蛋白这些区域的变构通讯，为复杂的 N 位交换可以直接从 H，N-HSQC 谱推断建立了一个范例。此外，在串联 SH2 和 γSA 结构中，分子动力学和 NMR 结果表明，PLCγ1 中的 Arg687Trp 突变（相当于 PLCγ2 中的癌症突变 Arg665Trp）扰乱了动态变构途径。这项结合实验和计算的研究揭示了一种罕见的多态动力学实例，涉及到一种动态变构过程，该过程在与疾病相关的突变的背景下受到调节。变构的影响和 cSH2-SH3 接头与 cSH2 的结合减弱应在对 PLCγ 调节的任何更全面的研究中加以考虑。
