PMID:21730060

Citation	Zhou, Y, Madej, MG, Guan, L, Nie, Y and Kaback, HR (2011) An early event in the transport mechanism of LacY protein: interaction between helices V and I. J. Biol. Chem. 286:30415-22
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.
Links	PubMed PMC3162401 Online version:10.1074/jbc.M111.268433
Keywords	Catalysis; Cross-Linking Reagents/chemistry; Cysteine/chemistry; Disulfides/chemistry; Electrochemistry/methods; Escherichia coli/metabolism; Escherichia coli Proteins/chemistry; Hydrogen Bonding; Lactose/chemistry; Ligands; Membrane Transport Proteins/chemistry; Mutation; Protein Binding; Protein Conformation; Protein Structure, Tertiary
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Annotations

Gene product	Qualifier	GO Term	Evidence Code	with/from	Aspect	Extension	Notes	Status
ECOLX:D0UXZ4		GO:0015155 : lactose transmembrane transporter activity	ECO:0000315:		F		Figure 2 shows significant lactose transmembrane transport activity of double Cys mutants in RSO membrane vesicles	complete CACAO 4898
ECOLX:D0UXZ4	enables	GO:0015155: lactose transmembrane transporter activity	ECO:0000315: mutant phenotype evidence used in manual assertion		F		Seeded From UniProt	complete

References

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