Thirumurugan Kavitha, Sakamoto Takeshi, Hammer John A, Sellers James R, Knight Peter J
Institute of Molecular and Cellular Biology, and Astbury Centre for Structural Molecular Biology, University of Leeds, Leeds LS2 9JT, UK.
Nature. 2006 Jul 13;442(7099):212-5. doi: 10.1038/nature04865.
Myosin 5 is a two-headed motor protein that moves cargoes along actin filaments. Its tail ends in paired globular tail domains (GTDs) thought to bind cargo. At nanomolar calcium levels, actin-activated ATPase is low and the molecule is folded. Micromolar calcium concentrations activate ATPase and the molecule unfolds. Here we describe the structure of folded myosin and the GTD's role in regulating activity. Electron microscopy shows that the two heads lie either side of the tail, contacting the GTDs at a lobe of the motor domain (approximately Pro 117-Pro 137) that contains conserved acidic side chains, suggesting ionic interactions between motor domain and GTD. Myosin 5 heavy meromyosin, a constitutively active fragment lacking the GTDs, is inhibited and folded by a dimeric GST-GTD fusion protein. Motility assays reveal that at nanomolar calcium levels heavy meromyosin moves robustly on actin filaments whereas few myosins bind or move. These results combine to show that with no cargo, the GTDs bind in an intramolecular manner to the motor domains, producing an inhibited and compact structure that binds weakly to actin and allows the molecule to recycle towards new cargoes.
肌球蛋白5是一种双头驱动蛋白,可沿着肌动蛋白丝移动货物。其尾部末端是成对的球状尾部结构域(GTD),被认为可结合货物。在纳摩尔钙水平下,肌动蛋白激活的ATP酶活性较低,分子处于折叠状态。微摩尔钙浓度会激活ATP酶,分子展开。在此,我们描述了折叠态肌球蛋白的结构以及GTD在调节活性中的作用。电子显微镜显示,两个头部位于尾部两侧,在运动结构域的一个叶(约Pro 117 - Pro 137)处与GTD接触,该叶含有保守的酸性侧链,表明运动结构域与GTD之间存在离子相互作用。肌球蛋白5重酶解肌球蛋白是一个缺乏GTD的组成型活性片段,被二聚体GST - GTD融合蛋白抑制并折叠。运动分析表明,在纳摩尔钙水平下,重酶解肌球蛋白在肌动蛋白丝上能强劲移动,而很少有肌球蛋白结合或移动。这些结果共同表明,在没有货物的情况下,GTD以分子内方式与运动结构域结合,产生一种抑制性的紧凑结构,该结构与肌动蛋白的结合较弱,使分子能够循环以寻找新的货物。