Maruta S, Saitoh J, Asakura T
Department of Bioengineering, Faculty of Engineering, Soka University, Hachioji, Tokyo 192-8577, Japan.
J Biochem. 2000 Feb;127(2):199-204. doi: 10.1093/oxfordjournals.jbchem.a022595.
Recent crystallographic studies have shown that smooth muscle myosin has three highly conserved unique loops, loop B (320-327), loop M (687-699), and loop N (125-134), similar to other myosins, skeletal muscle and dictyostelium myosins. We previously demonstrated that the effect of actin is mediated by a conformational change in one of the loops, loop M comprising amino acids 677 to 689 of skeletal muscle myosin [Maruta and Homma (1998) J. Biochem. 124, 528-533]. In the present study, in order to clarify the role of these smooth muscle myosin loops in energy transduction, we specifically labeled the loops with a fluorescent photoreactive ADP analogue, 3'-O-(N-methylanthraniloyl)-8-azido-ADP (Mant-8-N(3)-ADP), and then measured the fluorescent polarization. When Mant-8-N(3)-ADP was trapped by aluminium fluoride or vanadate into the ATPase site, Mant-8-N(3)-ADP was covalently incorporated into loop N (125-134). In contrast, Mant-8-N(3)-ADP trapped by beryllium fluoride was covalently incorporated into both loop M (687-699) and loop N (125-134) at an almost equimolar ratio. Actin binding to smooth muscle myosin S1 (SMO-S1) labeled at only loop N (125-134) increased the polarization due to the viscosity of actin. In contrast, S1 labeled at both loops N and M showed a much smaller increase in polarization. Our results indicate that the probe at loop M (687-699) of smooth muscle myosin moved to a less hindered region, suggesting that actin binding induces conformational changes at loop M (687-699) similar to those of the corresponding loop (677-689) in skeletal muscle myosin, as previously demonstrated in our laboratory.
最近的晶体学研究表明，平滑肌肌球蛋白有三个高度保守的独特环，环B（320 - 327）、环M（687 - 699）和环N（125 - 134），这与其他肌球蛋白，如骨骼肌和盘基网柄菌肌球蛋白类似。我们之前证明，肌动蛋白的作用是由其中一个环的构象变化介导的，环M包含骨骼肌肌球蛋白的677至689位氨基酸[Maruta和Homma（1998年）《生物化学杂志》124卷，528 - 533页]。在本研究中，为了阐明这些平滑肌肌球蛋白环在能量转导中的作用，我们用一种荧光光反应性ADP类似物3'-O-（N-甲基邻氨基苯甲酰基）-8-叠氮基-ADP（Mant-8-N(3)-ADP）对这些环进行特异性标记，然后测量荧光偏振。当Mant-8-N(3)-ADP被氟化铝或钒酸盐捕获到ATP酶位点时，Mant-8-N(3)-ADP共价结合到环N（125 - 134）中。相反，被氟化铍捕获的Mant-8-N(3)-ADP以几乎等摩尔的比例共价结合到环M（687 - 699）和环N（125 - 134）中。肌动蛋白与仅在环N（125 - 134）处标记的平滑肌肌球蛋白S1（SMO-S1）结合，由于肌动蛋白的粘性而增加了偏振。相反，在环N和M处都标记的S1偏振增加要小得多。我们的结果表明，平滑肌肌球蛋白环M（687 - 699）处的探针移动到了一个阻碍较小的区域，这表明肌动蛋白结合诱导环M（687 - 699）发生构象变化，类似于我们实验室之前证明的骨骼肌肌球蛋白中相应环（677 - 689）的构象变化。
