Surface-exposed positions in the transmembrane helices of the lactose permease of Escherichia coli determined by intermolecular thiol cross-linking

Abstract

bioenergetics ͉ transport ͉ membrane protein ͉ structure T he lactose permease of Escherichia coli (LacY) is a paradigm for membrane transport proteins that couple free energy stored in electrochemical ion gradients into solute concentration gradients (1-4). Thus, LacY catalyzes the coupled stoichiometric translocation of galactosides and H ϩ (lactose͞H ϩ symport), by using the free energy released from the downhill translocation of H ϩ to drive galactoside accumulation and vice versa. The protein has been solubilized from the membrane, purified in a completely functional state (reviewed in ref. 5) and shown to function as a monomer (6). LacY has 12 transmembrane helices with the N and C termini on the cytoplasmic face of the membrane ( Fig. 1; refs. 7-9).In a functional LacY mutant devoid of native Cys residues (Cys-less LacY), each residue has been replaced with Cys or other residues (reviewed in ref. 10). Systematic study of singleCys and other site-directed mutants has led to the identification of functionally essential residues (10) as well as a working model for the mechanism of lactose͞H ϩ symport (11,12). Analysis of the mutant library with a battery of site-directed biophysical and biochemical techniques has also led to the formulation of a helix-packing model of LacY ( Fig. 6; reviewed in ref. 13) In addition to other methods, intramolecular thiol crosslinking has been used for estimating helix packing, tilts, and ligand-induced conformational changes in LacY (12). While studying cross-linking of helix VI with homobifunctional thiol cross-linking agents in nonoverlapping, contiguous peptides corresponding to the N-and C-terminal halves of LacY (N 6 ͞C 6 split LacY), we observed (14) that certain paired-Cys mutants form C 6 ͞C 6 homodimers. The observation raised the possibility that such an approach might be useful for identifying residues in transmembrane helices that are surface exposed.In this study, homodimer formation induced by a homobifunctional thiol cross-linking agent 5 Å in length is observed with 9 single-Cys mutants in transmembrane helices of 250 mutants tested. Seven mutants are located at the cytoplasmic ends and 2 at periplasmic ends of transmembrane helices, and the positions are distributed around the periphery of the 12-helix bundle. The results are consistent with the current helix-packing model and suggest that Cys residues on helical surfaces exposed to the low dielectric of the membrane are unreactive. Moreover, two positions on opposite sides of the molecule cross-link at similar rates with sigmoid time courses, and cross-linking is markedly decreased at low temperature. The findings provide further evidence for the conclusion that LacY is a monomer, because homodimer formation appears to be a stochastic process involving random collisions within the plane of the membrane. Experimental ProceduresLacY Mutants. Construction of all single-Cys lacY mutants in plasmid pT7-5 has been described (15-24). Given mutants contain a 100-residue biotin acceptor domain (BAD) in the middle cy...

Lessons From Lactose Permease

“…The hydrophilic cavity is lined with helices I, II, IV, and V of the N-terminal domain and helices VII, VIII, X, and XI of the C-terminal domain. Helices III, VI, IX, and XII are embedded largely in the bilayer and not exposed to solvent, as suggested by Guan et al (31).…”

“…Finally, single-Cys replacement mutants in the transmembrane helices or loops of LacY dimerize in a stochastic manner (18,31,99). Moreover, the asymmetric unit of the LacY crystal is composed of an artificial dimer with two molecules oriented in opposite directions (2).…”

Probing the Structure of the Dimeric KtrB Membrane Protein

“…It has been reported that cross-linking easily occurs in the monomeric LacY, giving rise to an apparent dimer. Equivalent temperature experiments resulted in a reduction of the LacY dimer band by Ͼ90% (22). Fig.…”

“…4D). The low temperature was set below a phase transition of the E. coli lipid bilayer so that lateral diffusion within the membrane was decreased, resulting in a reduction of collisions between dimers (22,26). It has been reported that cross-linking easily occurs in the monomeric LacY, giving rise to an apparent dimer.…”

Role of the irreplaceable residues in the LacY alternating access mechanism

“…1). LacY has been solubilized from the membrane, purified to homogeneity in a highly functional state (2), and is structurally (3,4), as well as functionally (5), a monomer.…”