Thiol cross-linking of transmembrane domains IV and V in the lactose permease of Escherichia coli.
Glu126 (helix IV) and Arg144 (helix V) in the lactose permease of Escherichia coli are critical for substrate binding and transport, and the two residues are in close proximity and charge-paired. By using a functional permease construct with two tandem factor Xa protease sites in the cytoplasmic loop between helices IV and V, it is shown here that Cys residues in place of Glu126 and Arg144, as well as Ala122 and Val149, spontaneously form disulfide bonds in situ, indicating that this region of transmembrane domains IV and V is in the alpha-helical conformation. To determine if the local structure or environment is perturbed by the presence of an unpaired charge, either Glu126 or Arg144 or both were replaced with Ala, and cross-linking between the Cys pair Ala122-->Cys/Val149-->Cys was studied. Ala replacement for Arg144 causes a marked decrease in cross-linking, while Ala replacement for Glu126 alone or for both Glu126 and Arg144 has little effect. The data provide strong support for the argument that Glu126 and Arg144 are within close proximity and suggest that an unpaired carboxylate at position 126 causes a structural change at the interface between helices IV and V.
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Howard Hughes Medical Institute, Departments of Physiology and of Microbiology & Molecular Genetics, Molecular Biology Institute, University of California, Los Angeles, Los Angeles, California 90025-1662, USA.
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D000595:Amino Acid Sequence; D001120:Arginine / Q000737:chemistry / Q000235:genetics; D003432:Cross-Linking Reagents / Q000737:chemistry*; D003545:Cysteine / Q000737:chemistry / Q000235:genetics; D004926:Escherichia coli / Q000201:enzymology*; D029968:Escherichia coli Proteins; D015951:Factor Xa / Q000737:chemistry / Q000235:genetics; D018698:Glutamic Acid / Q000737:chemistry / Q000235:genetics; D008565:Membrane Proteins / Q000096:biosynthesis / Q000737:chemistry* / Q000235:genetics; D026901:Membrane Transport Proteins / Q000096:biosynthesis / Q000737:chemistry* / Q000235:genetics; D008969:Molecular Sequence Data; D009004:Monosaccharide Transport Proteins; D016297:Mutagenesis, Site-Directed; D010446:Peptide Fragments / Q000737:chemistry / Q000235:genetics; D017433:Protein Structure, Secondary / Q000235:genetics; D017434:Protein Structure, Tertiary / Q000235:genetics; D013438:Sulfhydryl Compounds / Q000737:chemistry*; D027981:Symporters
D003432:Cross-Linking Reagents; D029968:Escherichia coli Proteins; C050221:LacY protein, E coli; D008565:Membrane Proteins; D026901:Membrane Transport Proteins; D009004:Monosaccharide Transport Proteins; D010446:Peptide Fragments; D013438:Sulfhydryl Compounds; D027981:Symporters; D018698:Glutamic Acid; C021499:lactose permease; D001120:Arginine; D015951:Factor Xa; D003545:Cysteine