Mechanoenzymatics of titin kinase
Mechanoenzymatics of titin kinase
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DOI：
10.1073/pnas.0805034105
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发表时间：
2008-09-09
影响因子：
11.1
通讯作者：
Gautel, Mathias
中科院分区：
文献类型：
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作者：
Puchner, Elias M.;Alexandrovich, Alexander;Gautel, Mathias
对机械应力的生物反应需要应变感应分子，其机械诱导的构象变化会传递给介导细胞和组织特性变化的信号级联。在脊椎动物肌肉中，巨大的弹性蛋白肌联蛋白通过其在肌节M带的C末端激酶结构域（TK）参与应变感应，并有助于肌肉适应机械应变的变化。TK通过一种独特的双重自抑制机制受到调节，该机制由C末端调节尾实现，它阻断了ATP结合位点以及催化碱基的酪氨酸自抑制。为了能够接近ATP结合位点并使自抑制酪氨酸磷酸化，需要去除C末端自抑制尾。在此，我们利用基于原子力显微镜的单分子力谱、分子动力学模拟和酶学研究人类TK在应变诱导激活过程中的构象变化。我们表明，在结构肌联蛋白结构域展开之前，机械应变就激活了ATP结合，因此TK可作为一种生物力传感器。此外，我们确定了在低力作用下TK的自抑制被机械解除的步骤，这导致了辅底物ATP的结合，并使酶为随后的自磷酸化和底物周转做好准备。
Biological responses to mechanical stress require strain-sensing molecules, whose mechanically induced conformational changes are relayed to signaling cascades mediating changes in cell and tissue properties. In vertebrate muscle, the giant elastic protein titin is involved in strain sensing via its C-terminal kinase domain (TK) at the sarcomeric M-band and contributes to the adaptation of muscle in response to changes in mechanical strain. TK is regulated in a unique dual autoinhibition mechanism by a C-terminal regulatory tail, blocking the ATIP binding site, and tyrosine autoinhibition of the catalytic base. For access to the ATP binding site and phosphorylation of the autoinhibitory tyrosine, the C-terminal autoinhibitory tail needs to be removed. Here, we use AFM-based single-molecule force spectroscopy, molecular dynamics simulations, and enzymatics to study the conformational changes during strain-induced activation of human TK. We show that mechanical strain activates ATP binding before unfolding of the structural titin domains, and that TK can thus act as a biological force sensor. Furthermore, we identify the steps in which the autoinhibition of TK is mechanically relieved at low forces, leading to binding of the cosubstrate ATP and priming the enzyme for subsequent autophosphorylation and substrate turnover.