Journal
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA
Volume 112, Issue 29, Pages 9004-9009Publisher
NATL ACAD SCIENCES
DOI: 10.1073/pnas.1509854112
Keywords
X-ray structure; membrane protein; transport; major facilitator superfamily; conformational change
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Funding
- University of California Office of the President, Multicampus Research Programs and Initiatives Grant [MR-15-328599]
- Sandler Foundation
- National Institutes of Health [DK069463]
- National Science Foundation [MCB-1129551, R37GM024485]
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The X-ray crystal structure of a conformationally constrained mutant of the Escherichia coli lactose permease (the LacY double-Trp mutant Gly-46 -> Trp/Gly-262 -> Trp) with bound p-nitrophenyl-alpha-D-galactopyranoside (alpha-NPG), a high-affinity lactose analog, is described. With the exception of Glu-126 (helix IV), side chains Trp-151 (helix V), Glu-269 (helix VIII), Arg-144 (helix V), His-322 (helix X), and Asn-272 (helix VIII) interact directly with the galactopyranosyl ring of alpha-NPG to provide specificity, as indicated by biochemical studies and shown directly by X-ray crystallography. In contrast, Phe-20, Met-23, and Phe-27 (helix I) are within van der Waals distance of the benzyl moiety of the analog and thereby increase binding affinity nonspecifically. Thus, the specificity of LacY for sugar is determined solely by side-chain interactions with the galactopyranosyl ring, whereas affinity is increased by nonspecific hydrophobic interactions with the anomeric substituent.
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