MUTATIONS OF AN ACTIN BINDING LOOP IN HUMAN NONMUSCLE MYOSIN IIA

肌球蛋白表面环(氨基酸391-404)是 被认为是肌动蛋白的重要结合部位。在人类中 β-心肌肌球蛋白，它含有残基(Arg-403)，当 突变为Glu或Trp可导致肥厚型心肌病 (HCM)。棘阿米巴、棘阿米巴和肌球蛋白 和肌球蛋白VI类成员，这个环中的残基之一是 一成不变的是一串或三串。这种残基需要磷酸化。 对棘阿米巴和肌球蛋白I的活性几乎 所有其他肌球蛋白，这个残留物要么是谷氨酸，要么是天冬氨酸，表明 这个位置的负电荷对活动很重要。至 研究了这个循环的功能，我们使用了站点定向 重链肌球蛋白的诱变及杆状病毒表达 人非肌肉肌球蛋白IIA(HMM)样片段的研究 这个环路的突变。野生型表达的隐马尔可夫模型具有类似的 从非肌肉肌球蛋白IIA分离制备HMM的性质 来自人类的血小板。Arg393Gln突变(相当于 人β-心脏Arg403Gln HCM相关突变 肌肉肌球蛋白)基本上不影响肌动蛋白激活的 表达产物的镁ATPase活性或体外运动活性 嗯--像碎片。三种不同的突变，一种Asp399Lys 反转这个部位负电荷的突变，一个 Asp399Ala突变，消除了负电荷和 去除残基393-402的缺失突变都会降低 V-max增加8-10倍，对KATPase影响不大，降低 体外运动率是原来的2-3倍。这些数据支持 负电荷在这个位置的重要作用，但请注意 它对酶的活性并不重要。

A myosin surface loop (amino acids 391-404) is postulated to be an important binding site for actin. In human beta-cardiac myosin, it contains a residue (Arg-403) that when mutated to a Glu or Trp causes hypertrophic cardiomyopathy (HCM). In Acanthamoeba, Dicytostelium and Aspergillus myosin Is and members of myosin class VI, one of the residues in this loop is invariably a Ser or a Thr. Phosphorylation of this residue is required for activity of Acanthamoeba and Dicytostelium myosin I. In almost all other myosins, this residue is either a Glu or an Asp, suggesting that a negative charge at this location is important for activity. To study the function of this loop, we have used site-directed mutagenesis and baculovirus expression of an heavy meromyosin (HMM)-like fragment of human nonmuscle myosin IIA to study mutations of this loop. The wild-type expressed HMM has similar properties to HMM prepared from nonmuscle myosin IIA isolated from human platelets. An Arg393Gln mutation (equivalent to the Arg403Gln HCM-associated mutation in human beta-cardiac muscle myosin) has essentially no effect on the actin-activated MgATPase activity or in vitro motility activity of the expressed HMM-like fragment. Three different mutations, an Asp399Lys mutation which reverses the negative charge at this site, an Asp399Ala mutation which eliminates the negative charge and a deletion mutation which removes residues 393-402 all decrease the V-max by 8-10 fold with little effect on the KATPase and decrease the rate of in vitro motility by a factor of 2-3. These data support an important role for the negative charge at this location, but show that it is not critical to enzymatic activity.