Journal
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA
Volume 115, Issue 35, Pages 8769-8774Publisher
NATL ACAD SCIENCES
DOI: 10.1073/pnas.1801774115
Keywords
Major Facilitator Superfamily; X-ray structure; membrane protein; transport; conformational change
Categories
Funding
- University of California Office of the President, Multicampus Research Programs and Initiatives [MR-15-328599]
- Sandler Foundation
- National Institute of Health [R01 GM024485]
- NIH [GM120043]
- National Science Foundation [MCB1747705]
- INSTRUCT-ERIC
- Research Foundation-Flanders (FWO)
- NATIONAL INSTITUTE OF GENERAL MEDICAL SCIENCES [R01GM120043, R01GM024485] Funding Source: NIH RePORTER
Ask authors/readers for more resources
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 (LacY(WW)) 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(WW), the high-affinity substrate analog 4-nitrophenyl-alpha-D-galactoside (NPG), and Nb9047 at 3-angstrom 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(WW), which implicates dynamic flexibility of the Nb-LacY(WW) complex. Nb9047-binding neither changes the overall structure of LacY(WW) 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(WW)/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.
Authors
I am an author on this paper
Click your name to claim this paper and add it to your profile.
Reviews
Recommended
No Data Available