PMID:11771965

Citation	Zhang, W, Guan, L and Kaback, HR (2002) Helices VII and X in the lactose permease of Escherichia coli: proximity and ligand-induced distance changes. J. Mol. Biol. 315:53-62
Abstract	By using functional lactose permease devoid of native Cys residues with a discontinuity in the periplasmic loop between helices VII and VIII (N(7)/C(5) split permease), cross-linking between engineered paired Cys residues in helices VII and X was studied with the homobifunctional, thiol-specific cross-linkers 1,1-methanediyl bismethanethiosulfonate (3 A), N,N'-o- phenylenedimaleimide (6 A) and N,N'-p-phenylenedimaleimide (10 A). Mutant Asp240-->Cys (helix VII)/Lys319-->Cys (helix X) cross-links most efficiently with the 3 A reagent, providing direct support for studies indicating that Asp240 and Lys319 are in close proximity and charge paired. Furthermore, cross-linking the two positions inactivates the protein. Other Cys residues more disposed towards the middle of helix VII cross-link to Cys residues in the approximate middle of helix X, while no cross-linking is evident with paired Cys residues at the periplasmic or cytoplasmic ends of these helices. Thus, helices VII and X are in close proximity in the middle of the membrane. In the presence of ligand, the distance between Cys residues at positions 240 (helice VII) and 319 (helix X) increases. In contrast, the distance between paired Cys residues more disposed towards the cytoplasmic face of the membrane decreases in a manner suggesting that ligand binding induces a scissors-like movement between the two helices. The results are consistent with a recently proposed mechanism for lactose/H(+) symport in which substrate binding induces a conformational change between helices VII and X, during transfer of H(+) from His322 (helix X)/Glu269 (helix VIII) to Glu325 (helix X).
Links	PubMed Online version:10.1006/jmbi.2001.5206
Keywords	Amino Acid Sequence; Amino Acid Substitution/genetics; Binding Sites; Biological Transport, Active; Cell Membrane/metabolism; Cross-Linking Reagents/metabolism; Cysteine/chemistry; Cysteine/genetics; Cysteine/metabolism; Disulfides/chemistry; Disulfides/metabolism; Escherichia coli/cytology; Escherichia coli/enzymology; Escherichia coli/genetics; Escherichia coli Proteins; Lactose/metabolism; Ligands; Membrane Transport Proteins/chemistry; Membrane Transport Proteins/genetics; Membrane Transport Proteins/metabolism; Models, Molecular; Molecular Sequence Data; Monosaccharide Transport Proteins; Mutation/genetics; Protein Structure, Secondary; Symporters
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