Department of Biochemistry and Biophysics, University of California, San Francisco, CA 94158.
Department of Physiology, University of California, Los Angeles, CA 90095.
Proc Natl Acad Sci U S A. 2018 Aug 28;115(35):8769-8774. doi: 10.1073/pnas.1801774115. Epub 2018 Aug 14.
The lactose permease of (LacY), a dynamic polytopic membrane transport protein, catalyzes galactoside/H symport and operates by an alternating access mechanism that exhibits multiple conformations, the distribution of which is altered by sugar-binding. Camelid nanobodies were made against a double-mutant Gly46 → Trp/Gly262 → Trp (LacY) that produces an outward-open conformation, as opposed to the cytoplasmic open-state crystal structure of WT LacY. Nanobody 9047 (Nb9047) stabilizes WT LacY in a periplasmic-open conformation. Here, we describe the X-ray crystal structure of a complex between LacY, the high-affinity substrate analog 4-nitrophenyl-α-d-galactoside (NPG), and Nb9047 at 3-Å resolution. The present crystal structure demonstrates that Nb9047 binds to the periplasmic face of LacY, primarily to the C-terminal six-helical bundle, while a flexible loop of the Nb forms a bridge between the N- and C-terminal halves of LacY across the periplasmic vestibule. The bound Nb partially covers the vestibule, yet does not affect the on-rates or off-rates for the substrate binding to LacY, which implicates dynamic flexibility of the Nb-LacY complex. Nb9047-binding neither changes the overall structure of LacY with bound NPG, nor the positions of side chains comprising the galactoside-binding site. The current NPG-bound structure exhibits a more occluded periplasmic vestibule than seen in a previous structure of a (different Nb) apo-LacY/Nb9039 complex that we argue is caused by sugar-binding, with major differences located at the periplasmic ends of transmembrane helices in the N-terminal half of LacY.
（LacY）的乳糖通透酶是一种动态的多跨膜转运蛋白，催化半乳糖苷/H 共转运，通过交替访问机制发挥作用，该机制表现出多种构象，其分布通过糖结合而改变。针对产生外向开放构象的双突变 Gly46→Trp/Gly262→Trp（LacY），骆驼科纳米体被制成。与 WT LacY 的细胞质开放状态晶体结构相反。纳米体 9047（Nb9047）将 WT LacY 稳定在周质开放构象中。在这里，我们描述了 LacY、高亲和力底物类似物 4-硝基苯基-α-d-半乳糖苷（NPG）和 Nb9047 复合物的 X 射线晶体结构，分辨率为 3Å。目前的晶体结构表明，Nb9047 结合到 LacY 的周质面，主要结合到 C 端六螺旋束，而 Nb 的柔性环在周质前庭形成 LacY 的 N 和 C 端半之间的桥。结合的 Nb 部分覆盖前庭，但不影响底物与 LacY 结合的进入率或退出率，这表明 Nb-LacY 复合物具有动态灵活性。Nb9047 结合既不改变结合 NPG 的 LacY 的整体结构，也不改变构成半乳糖结合位点的侧链位置。当前的 NPG 结合结构显示出比我们之前提出的由糖结合引起的不同 Nb 的 apo-LacY/Nb9039 复合物的结构更闭塞的周质前庭，主要差异位于 LacY N 端半的跨膜螺旋的周质末端。
