Interaction between residues Glu269 (helix VIII) and His322 (helix X) of the lactose permease of Escherichia coli is essential for substrate binding.
Site-directed and Cys-scanning mutagenesis of the lactose permease of Escherichia coli reveals that as few as four residues--Glu269 (helix VIII), Arg302 (helix IV), His322 (helix X), and Glu325 (helix X)--are irreplaceable for coupling substrate and H+ translocation. Interestingly, the four residues are in close physical proximity, Glu269 interacting with His322 and Arg302 with Glu325. In addition, the substrate translocation pathway is located close to the four residues at the interface between helices V and VIII. To investigate the importance of the four residues and their interactions for substrate binding, mutation Glu269-->Asp, Glu269-->Gln, Arg302-->Ala, Arg302-->Lys, His322-->Ala, His322-->Phe, Glu325-->Asp, or Glu325-->Gln was introduced into single-Cys148 permease, where the reactivity of Cys with 2-(4-maleimidoanilino)naphthalene-6-sulfonic acid (MIANS) is blocked by binding of substrate. The double mutants were purified, and the rates of MIANS labeling were measured in the absence or presence of beta-D-galactopyranosyl 1-thio-beta-D-galactopyranoside (TDG), lactose, or galactose at various concentrations. Remarkably, substrate binding by the Glu269 or His322 mutants is abolished or decreased dramatically, while binding by the Arg302 or Glu325 mutants is not altered. The observations are consistent with the notion that the interaction between Glu269 and His322 stabilizes the interface between helices V and VIII and thereby leads to binding of substrate.
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Howard Hughes Medical Institute, Department of Physiology, University of California, Los Angeles 90095, USA.
Kaback
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HR
D000817:Anilino Naphthalenesulfonates; D019921:Biotinylation; D004926:Escherichia coli / Q000201:enzymology; D029968:Escherichia coli Proteins; D005690:Galactose / Q000494:pharmacology; D005973:Glutamine / Q000235:genetics / Q000378:metabolism*; D006639:Histidine / Q000235:genetics / Q000378:metabolism*; D007785:Lactose / Q000494:pharmacology; D026901:Membrane Transport Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D009004:Monosaccharide Transport Proteins; D016297:Mutagenesis, Site-Directed; D011485:Protein Binding / Q000187:drug effects / Q000235:genetics; D017433:Protein Structure, Secondary; D013379:Substrate Specificity / Q000187:drug effects / Q000235:genetics; D013439:Sulfhydryl Reagents; D027981:Symporters; D013862:Thiogalactosides / Q000494:pharmacology
D000817:Anilino Naphthalenesulfonates; D029968:Escherichia coli Proteins; C050221:LacY protein, E coli; D026901:Membrane Transport Proteins; D009004:Monosaccharide Transport Proteins; D013439:Sulfhydryl Reagents; D027981:Symporters; D013862:Thiogalactosides; D005973:Glutamine; D006639:Histidine; C021310:2-(4'-maleimidylanilino)naphthalene-6-sulfonic acid; C031803:thiodigalactoside; C021499:lactose permease; D007785:Lactose; D005690:Galactose