Gating-associated conformational changes in the mechanosensitive channel MscL
Gating-associated conformational changes in the mechanosensitive channel MscL
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DOI：
10.1073/pnas.0709436105
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发表时间：
2008-03-11
影响因子：
11.1
通讯作者：
Sokabe, Masahiro
中科院分区：
文献类型：
--
作者：
Yoshimura, Keniiro;Usukura, Jiro;Sokabe, Masahiro
细菌细胞通过机械敏感通道MscL的开放来避免低渗休克的溶解。当通道打开时，MscL被认为在膜平面上膨胀，形成一个直径约为3-4纳米的大孔。在这里，我们开始分析无细胞MscL的封闭和开放结构。为此，我们表征了野生型MscL的功能和结构，以及该蛋白的突变形式（G22N MscL）自发地采用开放亚态。对重组脂质体的MscL进行膜片钳分析发现，野生型MscL仅被机械刺激激活，而G22N MscL则表现出向开放亚态的自发开放。与这些结果一致，Ca2+内流到G22N含mscl脂质体发生在没有机械刺激。化学交联的G22N MscL的电泳迁移速度比交联的野生型MscL慢，表明G22N MscL处于扩展形态。最后，使用低角度旋转阴影的电子显微镜显示G22N MscL中心存在孔隙。野生型MscL未检测到孔。而野生型MscL一端有突出，G22N MscL没有。羧基末端27个残基的缺失导致了突起的丧失和正常的多聚。野生型和G22N型MscL的结构表明，MscL的打开伴随着羧基末端的解离和孔的形成。
Bacterial cells avoid lysis in response to hypoosmotic shock through the opening of the mechanosensitive channel MscL. Upon channel opening, MscL is thought to expand in the plane of the membrane and form a large pore with an estimated diameter of 3-4 nm. Here, we set out to analyze the closed and open structure of cell-free MscL. To this end, we characterized the function and structure of wild-type MscL and a mutant form of the protein (G22N MscL) that spontaneously adopts an open substate. Patch-clamp analysis of MscL that had been reconstituted into liposomes revealed that wild-type MscL was activated only by mechanical stimuli, whereas G22N MscL displayed spontaneous opening to the open substate. In accord with these results, Ca2+ influx into G22N MscL-containing liposomes occurred in the absence of mechanical stimulation. The electrophoretic migration of chemically crosslinked G22N MscL was slower than that of cross-linked wild-type MscL, suggesting that G22N MscL is in an expanded form. Finally, electron microscopy using low-angle rotary shadowing revealed the presence of a pore at the center of G22N MscL. No pore could be detected in wild-type MscL. However, wild-type MscL possessed a protrusion at one end, which was absent in G22N MscL. The deletion of carboxyl-terminal 27 residues resulted in the loss of protrusion and proper multimerization. The structures of wild-type and G22N MscL reveal that the opening of MscL is accompanied by the dissociation of a carboxyl-terminal protrusion and pore formation.