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Proc Natl Acad Sci U S A. 2018 Aug 28;115(35):8769-8774. doi: 10.1073/pnas.1801774115. Epub 2018 Aug 14.

Crystal Structure of a ligand-bound LacY-Nanobody Complex.

Author information

1
Department of Biochemistry and Biophysics, University of California, San Francisco, CA 94158.
2
Department of Physiology, University of California, Los Angeles, CA 90095.
3
VIB-VUB Center for Structural Biology, VIB, 1050 Brussels, Belgium.
4
Structural Biology Brussels, Vrije Universiteit Brussels, 1050 Brussels, Belgium.
5
Department of Physiology, University of California, Los Angeles, CA 90095; rkaback@mednet.ucla.edu stroud@msg.ucsf.edu.
6
Department of Microbiology, Immunology and Molecular Genetics, University of California, Los Angeles, CA 90095.
7
Molecular Biology Institute, University of California, Los Angeles, CA 90095.
8
Department of Biochemistry and Biophysics, University of California, San Francisco, CA 94158; rkaback@mednet.ucla.edu stroud@msg.ucsf.edu.

Abstract

The lactose permease of Escherichia coli (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 (LacYWW) 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 LacYWW, 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 LacYWW, which implicates dynamic flexibility of the Nb-LacYWW complex. Nb9047-binding neither changes the overall structure of LacYWW 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-LacYWW/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.

KEYWORDS:

Major Facilitator Superfamily; X-ray structure; conformational change; membrane protein; transport

PMID:
30108145
PMCID:
PMC6126719
DOI:
10.1073/pnas.1801774115
[Indexed for MEDLINE]
Free PMC Article

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