MUTATIONS OF AN ACTIN BINDING LOOP IN HUMAN NONMUSCLE MYOSIN IIA

虽然有肌动蛋白和肌球蛋白的晶体， 肌球蛋白与肌动蛋白的相互作用还没有很好的理解。 那里 是生物化学或模拟的结构证据， 肌球蛋白的几个区域与肌动蛋白，包括肌球蛋白表面环， 在人类中以氨基酸391开始并以氨基酸404结束 基于与鸡同源性的非肌肉肌球蛋白IIA序列 骨骼肌肌球蛋白结晶。 这个循环被假定为 根据Holmes-Rayment-Milligan原理， 与肌动蛋白和肌球蛋白相互作用的模型。 在人类心肌肌球蛋白中， 它含有突变为Gln或Trp的残基（Arg-403）， 肥厚型心肌病（HCM）。 在阿米巴和网骨藻中 肌球蛋白I和肌球蛋白VI类的成员， 该环总是丝氨酸或苏氨酸。 在肌球蛋白I 分子中，这种Thr的磷酸化是活性所必需的。 在 几乎所有其他肌球蛋白，这个残基是Glu或Asp， 这表明在这个残基上的负电荷对于 活动 我们已经使用了定点诱变结合 杆状病毒表达人非肌肉IIA的HMM样片段。 我们突变了Arg-393（相当于心肌肌球蛋白中的HCM突变） Gln和Asp-399（相当于监管机构） 磷酸化/不变的带负电荷的残基）转化为Lys。 两 突变体和野生型HMM的磷酸化调节良好， 20 kDa调节轻链。 Arg 393 Gln突变导致25- 肌动蛋白激活的MgATPase的Vmax增加50%。 突变 并不影响肌动蛋白丝在体外滑动的速率， 运动性测定 Asp 399 Lys突变使Vmax降低8-10倍 和体外运动率的两倍，支持一个 负电荷在这个位置的重要作用。 既不 突变对所需的肌动蛋白量有显着影响， 半最大MgATP酶活性，两者都受到其20 kD轻链磷酸化。 目前，我们正在研究其他 这个循环的变化。

While there are crystals of actin and of myosin, the molecular details of the interaction of myosin with actin are not well understood. There is a biochemical or modeled structural evidence for interaction between several areas of myosin with actin including a myosin surface loop that begins with amino acid 391 and ends with amino acid 404 in the human nonmuscle myosin IIA sequence based upon homology with the chicken skeletal muscle myosin that was crystallized. This loop is postulated to make direct contact with actin based on the Holmes-Rayment-Milligan model for interaction with actin and myosin. In human cardiac myosin, it contains a residue (Arg-403) that is mutated to a Gln or Trp to cause hypertrophic cardiomyopathy (HCM). In Acanthamoeba and Dictyostelium myosin I's and members of the myosin VI class, one of the residues in this loop is invariably a serine or threonine. In the myosin I molecules, phosphorylation of this Thr is required for activity. In almost all other myosins, this residue is either a Glu or Asp, suggesting that a negative charge at this residue is important for activity. We have used site-directed mutagenesis coupled with baculovirus expression of an HMM-like fragment of human nonmuscle IIA. We mutated Arg-393 (equivalent to the HCM mutation in cardiac myosin) to a Gln and Asp-399 (equivalent to the regulatory phosphorylation/invariant negative charged residue) to a Lys. Both mutants and wild-type HMM were well regulated by phosphorylation of the 20 kDa regulatory light chain. The Arg393Gln mutation results in a 25- 50% increase in the Vmax of the actin-activated MgATPase. The mutation did not affect the rate of actin filament sliding in an in vitro motility assay. The Asp399Lys mutation decreased the Vmax by 8-10 fold and the rate of in vitro motility by a factor of two, supporting an important role for the negative charge at this location. Neither mutation had a significant effect on the amount of actin required for half-maximal MgATPase activity and both are well regulated by their 20 kD light chain phosphorylation. We are currently studying other mutations of this loop.