Engineered occluded apo-intermediate of LacY-猫眼课题宝

LacY 的工程化封闭脱辅基中间体

DOI：

10.1073/pnas.1816267115

发表时间：

2018

期刊：

Proceedings of the National Academy of Sciences

影响因子：

通讯作者：

中科院分区：

文献类型：

作者：

Smirnova, Irina;Kasho, Vladimir;Kaback, H. Ronald

乳糖通透酶的大肠杆菌（LacY）利用交替访问symport机制与多个构象中间体，但只有向内（细胞质）或向外（周质）的开放结构已被X射线晶体学的特点。它表明在这里与糖结合的研究，交联配对半胱氨酸取代整个封闭的细胞质腔稳定闭塞的构象与一个难以接近的糖结合位点。此外，在WT LacY中稳定具有容易接近的糖结合位点的周质开放构象异构体的纳米抗体（Nb）未能引起细胞质交联突变体变得可接近半乳糖苷，这表明周质腔是封闭的。这些结果是一致的紧密关联的周质两端在两对螺旋包含集群的小残基之间的N-和C-末端六螺旋束的同向转运体的包装界面。然而，在二硫键还原后，Nb显著增加半乳糖苷结合的速率，表明从周质不受限制地接近Nb表位和半乳糖苷结合位点。研究结果表明，交联的胞质双半胱氨酸突变体类似于一个封闭的载脂蛋白中间体的运输周期。

The lactose permease ofEscherichia coli(LacY) utilizes an alternating access symport mechanism with multiple conformational intermediates, but only inward (cytoplasmic)- or outward (periplasmic)-open structures have been characterized by X-ray crystallography. It is demonstrated here with sugar-binding studies that cross-linking paired-Cys replacements across the closed cytoplasmic cavity stabilize an occluded conformer with an inaccessible sugar-binding site. In addition, a nanobody (Nb) that stabilizes a periplasmic-open conformer with an easily accessible sugar-binding site in WT LacY fails to cause the cytoplasmic cross-linked mutants to become accessible to galactoside, showing that the periplasmic cavity is closed. These results are consistent with tight association of the periplasmic ends in two pairs of helices containing clusters of small residues in the packing interface between N- and C-terminal six-helix bundles of the symporter. However, after reduction of the disulfide bond, the Nb markedly increases the rate of galactoside binding, indicating unrestricted access to the Nb epitope and the galactoside-binding site from the periplasm. The findings indicate that the cross-linked cytoplasmic double-Cys mutants resemble an occluded apo-intermediate in the transport cycle.