Olivares Adrian O, Chang Wakam, Mooseker Mark S, Hackney David D, De La Cruz Enrique M
Department of Molecular Biophysics and Biochemistry, Yale University, New Haven, Connecticut 06520, USA.
J Biol Chem. 2006 Oct 20;281(42):31326-36. doi: 10.1074/jbc.M603898200. Epub 2006 Aug 18.
Calcium activates full-length myosin Va steady-state enzymatic activity and favors the transition from a compact, folded "off" state to an extended "on" state. However, little is known of how a head-tail interaction alters the individual actin and nucleotide binding rate and equilibrium constants of the ATPase cycle. We measured the effect of calcium on nucleotide and actin filament binding to full-length myosin Va purified from chick brains. Both heads of nucleotide-free myosin Va bind actin strongly, independent of calcium. In the absence of calcium, bound ADP weakens the affinity of one head for actin filaments at equilibrium and upon initial encounter. The addition of calcium allows both heads of myosin Va.ADP to bind actin strongly. Calcium accelerates ADP binding to actomyosin independent of the tail but minimally affects ATP binding. Although 18O exchange and product release measurements favor a mechanism in which actin-activated Pi release from myosin Va is very rapid, independent of calcium and the tail domain, both heads do not bind actin strongly during steady-state cycling, as assayed by pyrene actin fluorescence. In the absence of calcium, inclusion of ADP favors formation of a long lived myosin Va.ADP state that releases ADP slowly, even after mixing with actin. Our results suggest that calcium activates myosin Va by allowing both heads to interact with actin and exchange bound nucleotide and indicate that regulation of actin binding by the tail is a nucleotide-dependent process favored by linked conformational changes of the motor domain.
钙可激活全长肌球蛋白Va的稳态酶活性，并有利于其从紧密折叠的“关闭”状态转变为伸展的“开启”状态。然而，关于头尾相互作用如何改变ATP酶循环中单个肌动蛋白和核苷酸结合速率以及平衡常数，人们所知甚少。我们测定了钙对从鸡脑中纯化的全长肌球蛋白Va与核苷酸和肌动蛋白丝结合的影响。无核苷酸的肌球蛋白Va的两个头部均能强烈结合肌动蛋白，且与钙无关。在没有钙的情况下，结合的ADP在平衡状态以及初始接触时会减弱一个头部对肌动蛋白丝的亲和力。添加钙后，肌球蛋白Va·ADP的两个头部均能强烈结合肌动蛋白。钙可加速ADP与肌动球蛋白的结合，与尾部无关，但对ATP结合的影响极小。尽管18O交换和产物释放测量结果支持一种机制，即肌动蛋白激活的肌球蛋白Va释放Pi的过程非常迅速，与钙和尾部结构域无关，但通过芘肌动蛋白荧光测定，在稳态循环过程中，两个头部与肌动蛋白的结合并不强烈。在没有钙的情况下，加入ADP有利于形成一种寿命较长的肌球蛋白Va·ADP状态，即使与肌动蛋白混合后，该状态也会缓慢释放ADP。我们的结果表明，钙通过使两个头部与肌动蛋白相互作用并交换结合的核苷酸来激活肌球蛋白Va，并表明尾部对肌动蛋白结合的调节是一个核苷酸依赖性过程，受运动结构域的连锁构象变化所青睐。
