Title | Crystal Structure of a ligand-bound LacY-Nanobody Complex. |
Publication Type | Journal Article |
Year of Publication | 2018 |
Authors | Kumar, H., J. S. Finer-Moore, X. Jiang, I. Smirnova, V. Kasho, E. Pardon, J. Steyaert, R. H Kaback, and R. M. Stroud |
Journal | Proc Natl Acad Sci U S A |
Volume | 115 |
Issue | 35 |
Pagination | 8769-8774 |
Date Published | 2018 08 28 |
ISSN | 1091-6490 |
Abstract | 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. |
DOI | 10.1073/pnas.1801774115 |
Alternate Journal | Proc. Natl. Acad. Sci. U.S.A. |
PubMed ID | 30108145 |
PubMed Central ID | PMC6126719 |
Grant List | R01 GM024485 / GM / NIGMS NIH HHS / United States |
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