Physics Department, University at Buffalo, Buffalo, NY 14260, USA.
Biochemistry. 2012 Jun 26;51(25):5022-32. doi: 10.1021/bi300362a. Epub 2012 Jun 15.
In this study, we have performed a comprehensive structural investigation of three major biochemical states of a kinesin complexed with microtubule under the constraint of high-quality cryo-electron-microscopy (EM) maps. In addition to the ADP and ATP state which were captured by X-ray crystallography, we have also modeled the nucleotide-free or APO state for which no crystal structure is available. We have combined flexible fitting of EM maps with regular molecular dynamics simulations, hydrogen-bond analysis, and free energy calculation. Our APO-state models feature a subdomain rotation involving loop L2 and α6 helix of kinesin, and local structural changes in active site similar to a related motor protein, myosin. We have identified a list of hydrogen bonds involving key residues in the active site and the binding interface between kinesin and microtubule. Some of these hydrogen bonds may play an important role in coupling microtubule binding to ATPase activities in kinesin. We have validated our models by calculating the binding free energy between kinesin and microtubule, which quantitatively accounts for the observation of strong binding in the APO and ATP state and weak binding in the ADP state. This study will offer promising targets for future mutational and functional studies to investigate the mechanism of kinesin motors.
在这项研究中，我们利用高质量的冷冻电镜（cryo-electron microscopy，简称 EM）图谱，对与微管结合的驱动蛋白的三种主要生化状态进行了全面的结构研究。除了通过 X 射线晶体学捕获的 ADP 和 ATP 状态外，我们还为没有晶体结构的无核苷酸或 APO 状态建模。我们将 EM 图谱的灵活拟合与正则分子动力学模拟、氢键分析和自由能计算相结合。我们的 APO 状态模型具有涉及驱动蛋白的环 L2 和α6 螺旋的亚结构域旋转，以及与相关的肌球蛋白运动蛋白相似的活性位点的局部结构变化。我们确定了涉及活性位点和驱动蛋白与微管结合界面的关键残基的氢键列表。这些氢键中的一些可能在将微管结合与驱动蛋白的 ATP 酶活性偶联中发挥重要作用。我们通过计算驱动蛋白与微管之间的结合自由能来验证我们的模型，这定量地解释了在 APO 和 ATP 状态下的强结合以及在 ADP 状态下的弱结合的观察结果。这项研究将为未来的突变和功能研究提供有前途的目标，以研究驱动蛋白的机制。
