Yengo Christopher M, De la Cruz Enrique M, Safer Daniel, Ostap E Michael, Sweeney H Lee
University of Pennsylvania School of Medicine, Department of Physiology and Pennsylvania Muscle Institute, Philadelphia, Pennsylvania 19104, USA.
Biochemistry. 2002 Jul 2;41(26):8508-17. doi: 10.1021/bi015969u.
Myosin V is a molecular motor shown to move processively along actin filaments. We investigated the properties of the weak binding states of monomeric myosin V containing a single IQ domain (MV 1IQ) to determine if the affinities of these states are increased as compared to conventional myosin. Further, using a combination of non-hydrolyzable nucleotide analogues and mutations that block ATP hydrolysis, we sought to probe the states that are populated during ATP-induced dissociation of actomyosin. MV 1IQ binds actin with a K(d) = 4 microM in the presence of ATP gamma S at 50 mM KCl, which is 10-20-fold tighter than that of nonprocessive class II myosins. Mutations within the switch II region trapped MV 1IQ in two distinct M.ATP states with very different actin binding affinities (K(d) = 0.2 and 2 microM). Actin binding may change the conformation of the switch II region, suggesting that elements of the nucleotide binding pocket will be in a different conformation when bound to actin than is seen in any of the myosin crystal structures to date.
肌球蛋白V是一种分子马达，已被证明能沿肌动蛋白丝连续移动。我们研究了含有单个IQ结构域的单体肌球蛋白V（MV 1IQ）的弱结合状态的特性，以确定与传统肌球蛋白相比，这些状态的亲和力是否增加。此外，我们使用不可水解的核苷酸类似物和阻断ATP水解的突变体的组合，试图探究在ATP诱导的肌动球蛋白解离过程中存在的状态。在50 mM KCl存在ATPγS的情况下，MV 1IQ以K(d)=4 microM的亲和力结合肌动蛋白，这比非连续的II类肌球蛋白的亲和力高10 - 20倍。开关II区域内的突变将MV 1IQ捕获在两种不同的M.ATP状态中，这两种状态具有非常不同的肌动蛋白结合亲和力（K(d)=0.2和2 microM）。肌动蛋白结合可能会改变开关II区域的构象，这表明核苷酸结合口袋的元件在与肌动蛋白结合时将处于与迄今为止任何肌球蛋白晶体结构中所见不同的构象。
