Extracellular-regulated kinase 2 is activated by the enhancement of hinge flexibility.
Extracellular-regulated kinase 2 is activated by the enhancement of hinge flexibility.
复制标题
细胞外调节激酶 2 通过铰链灵活性的增强而被激活。
DOI：
10.1016/j.jmb.2014.02.011
复制
发表时间：
2014
影响因子：
5.6
通讯作者：
Ahn,NatalieG
中科院分区：
文献类型：
--
作者：
Sours,KevinM;Xiao,Yao;Ahn,NatalieG
蛋白质运动的基础构象和熵的贡献，酶催化，然而，相对知之甚少的方式，这发生。通过氢交换质谱法对丝裂原活化蛋白激酶ERK 2（细胞外调节蛋白激酶2）的研究表明，活化增强了N-和C-末端结构域之间接头的骨架灵活性，同时改变了核苷酸结合模式。在这里，我们解决的假设，增强骨架的灵活性铰链区内促进激酶激活。我们表明，铰链突变增强灵活性，促进与结构域运动一致的核苷酸结合模式的变化，而不需要磷酸化。它们还导致单磷酸化ERK 2的激活，这是一种通常无活性的形式。铰链突变绕过对pTyr的需要，但不是pThr，表明Tyr磷酸化控制铰链运动。在协议中，单磷酸化的pTyr增强铰链的灵活性和核苷酸的结合模式，通过氢交换质谱法测量。我们的研究结果表明，调节蛋白质运动的激酶激活的基础。我们的工作模型是，在ERK 2的结构域运动的限制是克服磷酸化在pTyr，这增加了铰链动力学，以促进催化位点的活性构象。
Protein motions underlie conformational and entropic contributions to enzyme catalysis; however, relatively little is known about the ways in which this occurs. Studies of the mitogen-activated protein kinase ERK2 (extracellular-regulated proteinkinase2) by hydrogen-exchange mass spectrometry suggest that activation enhances backbone flexibility at the linker between N- and C-terminal domains while altering nucleotide binding mode. Here, we address the hypothesis that enhanced backbone flexibility within the hinge region facilitates kinase activation. We show that hinge mutations enhancing flexibility promote changes in the nucleotide binding mode consistent with domain movement, without requiring phosphorylation. They also lead to the activation of monophosphorylated ERK2, a form that is normally inactive. The hinge mutations bypass the need for pTyr but not pThr, suggesting that Tyr phosphorylation controls hinge motions. In agreement, monophosphorylation of pTyr enhances both hinge flexibility and nucleotide binding mode, measured by hydrogen-exchange mass spectrometry. Our findings demonstrate that regulated protein motions underlie kinase activation. Our working model is that constraints to domain movement in ERK2 are overcome by phosphorylation at pTyr, which increases hinge dynamics to promote the active conformation of the catalytic site.