Vinogradova Maia V, Malanina Galina G, Reddy Vaka S, Reddy Anireddy S N, Fletterick Robert J
Department of Biochemistry/Biophysics, University of California, 600 16th Street GH S412E, San Francisco, CA 94107, USA.
J Struct Biol. 2008 Jul;163(1):76-83. doi: 10.1016/j.jsb.2008.04.004. Epub 2008 May 5.
Kinesins are molecular motors that power cell division and transport of various proteins and organelles. Their motor activity is driven by ATP hydrolysis and depends on interactions with microtubule tracks. Essential steps in kinesin movement rely on controlled alternate binding to and detaching from the microtubules. The conformational changes in the kinesin motors induced by nucleotide and microtubule binding are coordinated by structural elements within their motor domains. Loop L11 of the kinesin motor domain interacts with the microtubule and is implicated in both microtubule binding and sensing nucleotide bound to the active site of kinesin. Consistent with its proposed role as a microtubule sensor, loop L11 is rarely seen in crystal structures of unattached kinesins. Here, we report four structures of a regulated plant kinesin, the kinesin-like calmodulin binding protein (KCBP), determined by X-ray crystallography. Although all structures reveal the kinesin motor in the ATP-like conformation, its loop L11 is observed in different conformational states, both ordered and disordered. When structured, loop L11 adds three additional helical turns to the N-terminal part of the following helix alpha4. Although interactions with protein neighbors in the crystal support the ordering of loop L11, its observed conformation suggests the conformation for loop L11 in the microtubule-bound kinesin. Variations in the positions of other features of these kinesins were observed. A critical regulatory element of this kinesin, the calmodulin binding helix positioned at the C-terminus of the motor domain, is thought to confer negative regulation of KCBP. Calmodulin binds to this helix and inserts itself between the motor and the microtubule. Comparison of five independent structures of KCBP shows that the positioning of the calmodulin binding helix is not decided by crystal packing forces but is determined by the conformational state of the motor. The observed variations in the position of the calmodulin binding helix fit the regulatory mechanism previously proposed for this kinesin motor.
驱动蛋白是一种分子马达，为细胞分裂以及各种蛋白质和细胞器的运输提供动力。它们的马达活性由ATP水解驱动，并依赖于与微管轨道的相互作用。驱动蛋白运动的关键步骤依赖于与微管的可控交替结合和解离。核苷酸和微管结合诱导的驱动蛋白马达的构象变化由其马达结构域内的结构元件协调。驱动蛋白马达结构域的环L11与微管相互作用，并且与微管结合以及感知结合在驱动蛋白活性位点上的核苷酸都有关。与其作为微管传感器的推测作用一致，在未附着的驱动蛋白的晶体结构中很少见到环L11。在这里，我们报告了通过X射线晶体学确定的一种受调控的植物驱动蛋白——类驱动蛋白钙调蛋白结合蛋白（KCBP）的四种结构。尽管所有结构都显示驱动蛋白马达处于ATP样构象，但其环L11处于不同的构象状态，既有有序的也有无序的。当形成结构时，环L11在接下来的α4螺旋的N端部分增加了三个额外的螺旋圈。尽管与晶体中的蛋白质邻居的相互作用支持环L11的有序化，但其观察到的构象表明了微管结合的驱动蛋白中环L11的构象。观察到了这些驱动蛋白其他特征位置的变化。这种驱动蛋白的一个关键调节元件，即位于马达结构域C端的钙调蛋白结合螺旋，被认为赋予了KCBP负调控作用。钙调蛋白与这个螺旋结合并插入马达和微管之间。KCBP的五个独立结构的比较表明，钙调蛋白结合螺旋的定位不是由晶体堆积力决定的，而是由马达的构象状态决定的。观察到的钙调蛋白结合螺旋位置的变化符合先前为这种驱动蛋白马达提出的调节机制。
