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.
Highlights
Typical of many transport proteins from organisms as disparate as Archaea and Homo sapiens, the lactose permease of Escherichia coli (LacY), a member of the major facilitator superfamily [1], catalyzes the coupled, stoichiometric translocation of an Hϩ and a galactopyranoside
Crosslinking was performed in the absence or presence of the lactose homologue -D-galactopyranosyl 1-thio--D-galactopyranoside (TDG), and the effect on transport activity was tested as well
Site-directed thiol cross-linking in situ with mutants containing an engineered protease site is a powerful approach for estimating helix packing and probing ligand-induced conformational changes, as cross-linking reactions can be performed before and after interaction with substrate
Summary
9 and 24 –30), single molecule fluorescence resonance energy transfer [31], double electron-electron resonance [8], and Trp fluorescence quenching [32], demonstrate that an outward-facing conformation exists, but that a large periplasmic cavity must open and close for transport to occur [24] By this means, the cytoplasmic cavity closes with opening of a wide hydrophilic cavity on the periplasmic side of LacY, which allows exposure of sugar- and Hϩ-binding sites to either side of the membrane (the alternating access mechanism) (reviewed in Ref. 33). Site-directed mutagenesis experiments on the native Cys residues in LacY led to the isolation and characterization of a mutant with Gly in place of native Cys154 (Helix V) [38, 39] This single mutation completely changes the functional properties and physical characteristics of LacY [40]. Further evidence for this proposal is provided by the thiol cross-linking studies between helices V and I presented here
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