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Proc Natl Acad Sci U S A. 2015 Jul 21;112(29):9004-9. doi: 10.1073/pnas.1509854112. Epub 2015 Jul 8.

Structure of LacY with an α-substituted galactoside: Connecting the binding site to the protonation site.

Author information

1
Department of Biochemistry and Biophysics, University of California, San Francisco, CA 94158;
2
Department of Physiology and Department of Microbiology, Immunology, and Molecular Genetics, and Molecular Biology Institute, University of California, Los Angeles, CA 90095 rkaback@mednet.ucla.edu stroud@msg.ucsf.edu.
3
Department of Biochemistry and Biophysics, University of California, San Francisco, CA 94158; rkaback@mednet.ucla.edu stroud@msg.ucsf.edu.

Abstract

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-α-d-galactopyranoside (α-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 α-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.

KEYWORDS:

X-ray structure; conformational change; major facilitator superfamily; membrane protein; transport

PMID:
26157133
PMCID:
PMC4517220
DOI:
10.1073/pnas.1509854112
[Indexed for MEDLINE]
Free PMC Article

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