Department of Physiology, University of California, Los Angeles, CA 90095-7327.
Department of Physiology, University of California, Los Angeles, CA 90095-7327;
Proc Natl Acad Sci U S A. 2018 Dec 11;115(50):12716-12721. doi: 10.1073/pnas.1816267115. Epub 2018 Nov 26.
The lactose permease of (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.
（LacY）的乳糖通透酶利用多个构象中间产物的交替访问协同转运机制，但只有通过 X 射线晶体学才能对内向（细胞质）或外向（周质）开放结构进行特征描述。通过糖结合研究表明，跨封闭细胞质腔的配对半胱氨酸取代的交联可稳定一个具有不可接近的糖结合位点的闭塞构象。此外，纳米体（Nb）稳定 WT LacY 中的周质开放构象，其糖结合位点易于接近，但不能使细胞质交联突变体对半乳糖苷变得可及，表明周质腔是封闭的。这些结果与紧密相关的周质末端在两个对螺旋，其中包含小残基簇在包装接口之间 N -和 C -末端六螺旋束的协同转运体。然而，在二硫键还原后，Nb 显著增加了半乳糖苷结合的速率，表明从周质中不受限制地进入 Nb 表位和半乳糖苷结合位点。研究结果表明，交联的细胞质双半胱氨酸突变体类似于转运循环中的闭塞 apo 中间产物。
