Localization and Characterization of the Inhibitory Ca2+-binding Site of Physarum polycephalum Myosin II*
Localization and Characterization of the Inhibitory Ca2+-binding Site of Physarum polycephalum Myosin II*
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DOI：
10.1074/jbc.m304220200
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发表时间：
2003-07
影响因子：
4.8
通讯作者：
L. Farkas;A. Málnási-Csizmadia;A. Nakamura;K. Kohama;L. Nyitray
中科院分区：
文献类型：
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作者：
L. Farkas;A. Málnási-Csizmadia;A. Nakamura;K. Kohama;L. Nyitray
一种肌球蛋白II被认为是多头绒泡菌原质团中快速细胞质流动的驱动力。这种受调节的肌球蛋白在常规肌球蛋白中是独特的，被直接的Ca 2+结合抑制。在这里，我们报告，钙结合的第一EF-手的必需轻链（ELC）亚基的绒泡菌肌球蛋白。野生型和突变体轻链和调节结构域的流动透析实验揭示了一个单一的结合位点，表现出中等特异性的Ca 2+。与高等真核生物的调节轻链相反，调节轻链不能结合二价阳离子。虽然ELC的Ca 2+结合环具有典型序列，但在-z配位位置将谷氨酸替换为丙氨酸仅略微降低该位点的Ca 2+亲和力，表明Ca 2+配位不同于经典EF-手;即，特定的“闭合-开放”构象转变不会在ELC中响应于Ca 2+而发生。通过荧光实验检测结合位点微环境中Ca 2+和Mg 2+依赖的构象变化。瞬时动力学实验表明，Mg 2+的位移Ca 2+是快于细胞质流动方向的变化，因此，我们得出结论，Ca 2+抑制可以在生理条件下操作。通过比较绒泡菌的Ca ~（2+）位点和扇贝肌球蛋白的Ca ~（2+）开关，我们推测，尽管Ca ~（2+）结合对运动活性的作用相反，但这两种常规肌球蛋白可能具有共同的Ca ~（2+）调节结构基础。
A myosin II is thought to be the driving force of the fast cytoplasmic streaming in the plasmodium of Physarum polycephalum. This regulated myosin, unique among conventional myosins, is inhibited by direct Ca2+ binding. Here we report that Ca2+ binds to the first EF-hand of the essential light chain (ELC) subunit of Physarum myosin. Flow dialysis experiments of wild-type and mutant light chains and the regulatory domain revealed a single binding site that shows moderate specificity for Ca2+. The regulatory light chain, in contrast to regulatory light chains of higher eukaryotes, is unable to bind divalent cations. Although the Ca2+-binding loop of ELC has a canonical sequence, replacement of glutamic acid to alanine in the –z coordinating position only slightly decreased the Ca2+ affinity of the site, suggesting that the Ca2+ coordination is different from classical EF-hands; namely, the specific “closed-to-open” conformational transition does not occur in the ELC in response to Ca2+. Ca2+- and Mg2+-dependent conformational changes in the microenvironment of the binding site were detected by fluorescence experiments. Transient kinetic experiments showed that the displacement of Mg2+ by Ca2+ is faster than the change in direction of cytoplasmic streaming; therefore, we conclude that Ca2+ inhibition could operate in physiological conditions. By comparing the Physarum Ca2+ site with the well studied Ca2+ switch of scallop myosin, we surmise that despite the opposite effect of Ca2+ binding on the motor activity, the two conventional myosins could have a common structural basis for Ca2+ regulation.