Switch II Mutants Reveal Coupling between the Nucleotide- and Actin-Binding Regions in Myosin V
Switch II Mutants Reveal Coupling between the Nucleotide- and Actin-Binding Regions in Myosin V
复制标题
DOI：
10.1016/j.bpj.2012.04.025
复制
发表时间：
2012-06-06
影响因子：
3.4
通讯作者：
Yengo, Christopher M.
中科院分区：
文献类型：
--
作者：
Trivedi, Darshan V.;David, Charles;Yengo, Christopher M.
肌球蛋白和其他p环NTPases中的保守活性位点元件在核苷酸结合和水解中起关键作用；然而，这些机械酶之间的变构通讯机制仍未得到解决。在这项工作中，我们引入了E442A突变，该突变消除了开关I和开关II之间的盐桥，以及G440A突变，该突变消除了与开关II与ATP γ磷酸相互作用相关的主链氢键，进入肌凝蛋白V。我们使用荧光共振能量转移在mant标记的核苷酸或iaedans标记的肌动蛋白和flash标记的肌凝蛋白V之间分别检测核苷酸和肌动蛋白结合区域的构象。我们证明，在没有肌动蛋白的情况下，G440A和E442A突变体都以相似的亲和力结合ATP，并且只导致核苷酸结合袋（NBP）的构象发生轻微改变。在ADP和肌动蛋白存在的情况下，两种开关II突变体都会破坏封闭的NBP肌动球蛋白的形成。ADP的状态。G440A突变体也能阻止atp诱导的肌动蛋白结合间隙的打开。我们的研究结果表明，在肌动蛋白存在的情况下，开关II区对于稳定封闭的NBP构象至关重要，并且对于活性位点和肌动蛋白结合区之间的通信至关重要。
Conserved active-site elements in myosins and other P-loop NTPases play critical roles in nucleotide binding and hydrolysis; however, the mechanisms of allosteric communication among these mechanoenzymes remain unresolved. In this work we introduced the E442A mutation, which abrogates a salt-bridge between switch I and switch II, and the G440A mutation, which abolishes a main-chain hydrogen bond associated with the interaction of switch II with the gamma phosphate of ATP, into myosin V. We used fluorescence resonance energy transfer between mant-labeled nucleotides or IAEDANS-labeled actin and FlAsH-labeled myosin V to examine the conformation of the nucleotide-and actin-binding regions, respectively. We demonstrate that in the absence of actin, both the G440A and E442A mutants bind ATP with similar affinity and result in only minor alterations in the conformation of the nucleotide-binding pocket (NBP). In the presence of ADP and actin, both switch II mutants disrupt the formation of a closed NBP actomyosin. ADP state. The G440A mutant also prevents ATP-induced opening of the actin-binding cleft. Our results indicate that the switch II region is critical for stabilizing the closed NBP conformation in the presence of actin, and is essential for communication between the active site and actin-binding region.