title: An early event in the transport mechanism of LacY protein: interaction between helices V and I.
abstract: Helix V in LacY, which abuts and crosses helix I in the N-terminal helix bundle of LacY, contains Arg(144) and Trp(151), two residues that play direct roles in sugar recognition and binding, as well as Cys(154), which is important for conformational flexibility. In this study, paired Cys replacement mutants in helices V and I were strategically constructed with tandem factor Xa protease cleavage sites in the loop between the two helices to test cross-linking. None of the mutants form disulfides spontaneously; however, three mutants (Pro(28) → Cys/Cys(154), Pro(28) → Cys/Val(158) → Cys, and Phe(29) → Cys/Val(158) → Cys) exhibit cross-linking after treatment with copper/1,10-phenanthroline (Cu/Ph) or 1,1-methanediyl bismethanethiosulfonate ((MTS)(2)-1), 3-4 Å), and cross-linking is quantitative in the presence of ligand. Remarkably, with one mutant, complete cross-linking with (MTS)(2)-1 has no effect on lactose transport, whereas quantitative disulfide cross-linking catalyzed by Cu/Ph markedly inhibits transport activity. The findings are consistant with a number of previous conclusions suggesting that sugar binding to LacY causes a localized scissors-like movement between helices V and I near the point where the two helices cross in the middle of the membrane. This ligand-induced movement may act to initiate the global conformational change resulting from sugar binding.
authors: Departments of Physiology, University of California, Los Angeles, California 90095-1662.
authors: Yonggang
authors: Y
authors: Zhou
authors: Departments of Physiology, University of California, Los Angeles, California 90095-1662.
authors: M Gregor
authors: MG
authors: Madej
authors: Departments of Physiology, University of California, Los Angeles, California 90095-1662.
authors: Lan
authors: L
authors: Guan
authors: Departments of Physiology, University of California, Los Angeles, California 90095-1662.
authors: Yiling
authors: Y
authors: Nie
authors: Departments of Physiology, University of California, Los Angeles, California 90095-1662; Microbiology, Immunology and Molecular Genetics, University of California, Los Angeles, California 90095-1662; Molecular Biology Institute, University of California, Los Angeles, California 90095-1662. Electronic address: rkaback@mednet.ucla.edu.
authors: H Ronald
authors: HR
authors: Kaback
mesh_terms: D002384:Catalysis; D003432:Cross-Linking Reagents / Q000737:chemistry; D003545:Cysteine / Q000737:chemistry; D004220:Disulfides / Q000737:chemistry; D004563:Electrochemistry / Q000379:methods; D004926:Escherichia coli / Q000378:metabolism*; D029968:Escherichia coli Proteins / Q000737:chemistry; D006860:Hydrogen Bonding; D007785:Lactose / Q000737:chemistry; D008024:Ligands; D026901:Membrane Transport Proteins / Q000737:chemistry*; D009154:Mutation; D011485:Protein Binding; D011487:Protein Conformation; D017434:Protein Structure, Tertiary
chemical_list: D003432:Cross-Linking Reagents; D004220:Disulfides; D029968:Escherichia coli Proteins; D008024:Ligands; D026901:Membrane Transport Proteins; C021499:lactose permease; D007785:Lactose; D003545:Cysteine