Department of Physiology and ‡Department of Microbiology, Immunology & Molecular Genertics, §Molecular Biology Institute, University of California Los Angeles , Los Angeles, California 90095, United States.
Biochemistry. 2013 Sep 17;52(37):6568-74. doi: 10.1021/bi401026d. Epub 2013 Aug 30.
The lactose permease from Escherichia coli (LacY) is a galactoside/H(+) symporter that catalyzes the coupled stoichiometric transport of a sugar and an H(+) across the cytoplasmic membrane. X-ray crystal structures of WT LacY and the conformationally restricted mutant Cys154→Gly exhibit an inward-facing conformation with a tightly sealed periplasmic side and a deep central cleft or cavity open to the cytoplasm. Although the crystal structures may give the impression that LacY is a rigid molecule, multiple converging lines of evidence demonstrate that galactoside binding to WT LacY induces reciprocal opening and closing of periplasmic and cytoplasmic cavities, respectively. By this means, the sugar- and H(+)-binding sites in the middle of the molecule are exposed alternatively to either side of the membrane. In contrast to the crystal structure, biochemical/biophysical studies with mutant Cys154→Gly show that the periplasmic side is paralyzed in an open-outward conformation. In this study, a rigid, funnel-shaped, maleimide-containing molecule was used to probe the periplasmic cavity of a pseudo-WT and the Cys154→Gly mutant by site-directed alkylation. The findings provide strong support for previous observations and indicate further that the external opening of the periplasmic cleft in the mutant is patent to the extent of at least 8.5 Å in the absence of sugar or about half that of the WT cavity with bound galactoside.
大肠杆菌的乳糖通透酶（LacY）是一种半乳糖苷/H(+)协同转运蛋白，可催化糖和 H(+)的协同转运穿过细胞质膜。野生型 LacY 和构象受限的突变体 Cys154→Gly 的 X 射线晶体结构显示出一种面向内的构象，其周质侧紧密密封，中央深裂或腔隙朝向细胞质开放。尽管晶体结构可能给人一种 LacY 是刚性分子的印象，但多方面的证据表明，半乳糖苷与野生型 LacY 的结合分别诱导周质腔和细胞质腔的相互开放和关闭。通过这种方式，分子中部的糖和 H(+)结合位点交替暴露于膜的两侧。与晶体结构相反，对突变体 Cys154→Gly 的生化/生物物理研究表明，周质侧在向外张开的构象中瘫痪。在这项研究中，刚性的、漏斗形的、含有马来酰亚胺的分子被用于通过定点烷基化来探测伪 WT 和 Cys154→Gly 突变体的周质腔。这些发现为以前的观察结果提供了有力支持，并进一步表明，在没有糖的情况下，突变体中周质裂隙的外部开口至少有 8.5Å 的开度，而与结合的半乳糖苷相比，WT 腔隙的开度约为其一半。
