title: Structure of LacY with an α-substituted galactoside: Connecting the binding site to the protonation site.
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.
authors.affiliation: Department of Biochemistry and Biophysics, University of California, San Francisco, CA 94158;
authors.forename: Hemant
authors.initials: H
authors.lastname: Kumar
authors.affiliation: Department of Biochemistry and Biophysics, University of California, San Francisco, CA 94158;
authors.forename: Janet S
authors.initials: JS
authors.lastname: Finer-Moore
authors.affiliation: 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.
authors.forename: H Ronald
authors.initials: HR
authors.lastname: Kaback
authors.affiliation: Department of Biochemistry and Biophysics, University of California, San Francisco, CA 94158; rkaback@mednet.ucla.edu stroud@msg.ucsf.edu.
authors.forename: Robert M
authors.initials: RM
authors.lastname: Stroud
mesh_terms: D001665:Binding Sites; D018360:Crystallography, X-Ray; D004926:Escherichia coli / Q000201:enzymology*; D029968:Escherichia coli Proteins / Q000737:chemistry* / Q000378:metabolism*; D008024:Ligands; D008958:Models, Molecular; D009004:Monosaccharide Transport Proteins / Q000737:chemistry* / Q000378:metabolism*; D009598:Nitrophenylgalactosides / Q000378:metabolism*; D017433:Protein Structure, Secondary; D011522:Protons; D055672:Static Electricity; D013379:Substrate Specificity; D027981:Symporters / Q000737:chemistry* / Q000378:metabolism*
chemical_list: D029968:Escherichia coli Proteins; C050221:LacY protein, E coli; D008024:Ligands; D009004:Monosaccharide Transport Proteins; D011522:Protons; D027981:Symporters; D009598:Nitrophenylgalactosides; C059200:4-nitrophenylgalactoside
keywords: X-ray structure; conformational change; major facilitator superfamily; membrane protein; transport