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. 2002 Mar 19;99(6):3475–3480. doi: 10.1073/pnas.052703699

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

Lan Guan 1, Franklin D Murphy 1, H Ronald Kaback 1,*
PMCID: PMC122548  PMID: 11904412

Abstract

Intermolecular thiol cross-linking was used to determine surface-exposed positions in 250 lactose permease mutants containing single-Cys replacements in each transmembrane helix. Significant cross-linking of monomers to produce homodimers is observed in nine mutants with a 5-Å-long cross-linking agent containing bis-methane thiosulfonate reactive groups [position 78 (helix III); positions 185, 186, and 187 (helix VI); positions 263, 275, and 278 (helix VIII); and positions 308 (helix IX) and 398 (helix XII)]. The results are consistent with a current helix-packing model of the permease. Seven of the nine mutants that exhibit intermolecular cross-linking are located at or near the cytoplasmic ends of transmembrane helices; two are near periplasmic ends. The results suggest that only those Cys replacements accessible from the aqueous phase and not from the hydrophobic core of the membrane are susceptible to cross-linking because of the much higher reactivity of the thiolate anion relative to the thiol. Single-Cys mutants at positions 278 (helix VIII) and 398 (helix XII), which are located in opposite sides of the 12-helix bundle, exhibit similar rates of cross-linking with sigmoid kinetics. Furthermore, cross-linking is markedly decreased at 0°C, suggesting that lateral diffusion of the permease within the plane of the membrane is important for intermolecular cross-linking. The findings confirm previous observations indicating that intermolecular cross-linking is a stochastic process resulting from random collisions and support a number of other lines of evidence that lactose permease is a monomer.

Keywords: bioenergetics‖transport‖membrane protein‖structure

The 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+ (lactose/H+ symport), by using the free energy released from the downhill translocation of H+ 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).

Figure 1.

Figure 1

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Secondary structure model of LacY showing positions of Cys-replacement mutants used. Helix-loop boundaries were approximated by single amino acid deletion analysis (36, 37). Residues in transmembrane helices are shown in rectangles. Shaded rectangles or single residues enclosed in shaded circles highlight the single-Cys mutants tested in this study. Residues enclosed in dark circles exhibit homodimer formation in the presence of given homobifunctional cross-linking agents. Encircled residues in periplasmic loops represent positions where Cys replacement resulting in spontaneous disulfide formation (46).

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 single-Cys 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 lactose/H+ 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)

Figure 6.

Figure 6

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Helix packing model of LacY showing positions where Cys replacement leads to homodimer formation. Shown is the current helix packing model derived from over 100 distance constraints by M. Girvin (48; see ref. 12) with the native side chains at the positions that form homodimers when replaced with a Cys residue.

In addition to other methods, intramolecular thiol cross-linking 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 (N6/C6 split LacY), we observed (14) that certain paired-Cys mutants form C6/C6 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 Procedures

LacY 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 cytoplasmic loop (loop VI–VII) or at the C terminus. Construction of the LacY/LacY tandem fusion protein has also been described (6).

Expression of LacY and Membrane Preparation.

E. coli T184 [lacI+O+Z−Y− (A) rpsL met− thr− recA hsdM hsdR/F′ lacIq O+ZD118 (Y+ A+)] (25) was transformed with plasmid pT7-5 encoding the cassette lacY gene with given single-Cys mutants. Cells were grown at 37°C in Luria–Bertani broth containing 100 μg/ml ampicillin and 0.1 mM isopropyl 1-thio-β,D-galactopyranoside. Overnight cultures were harvested by centrifugation, washed with 20 mM Tris⋅HCl (pH 7.4)/5.0 mM EDTA, suspended in the same buffer, and disrupted by sonification. After centrifugation at 20,000 × gmax for 15 min at 4°C to remove unbroken cells, membranes were harvested from the supernatant by centrifugation at 440,000 × gmax for 10 min at 4°C. The supernatant was discarded, and the pellet was suspended in 20 mM Tris⋅HCl (pH 7.4) at a protein concentration of about 2 mg/ml. Total membrane protein was assayed with the MicroBCA Kit (Pierce) by using BSA as the standard.

Cross-Linking.

Unless stated otherwise, cross-linking reactions were carried out at room temperature for 1 h in the presence of the homobifunctional cross-linking agent 1,3 propanediyl bismethanethiosulfonate [MTS-3-MTS (5 Å); Toronto Research Chemicals, Downsview, ON, Canada] at a final concentration of 0.05 mM. When 1,1-methanediyl bismethanethiosulfonate [MTS-1-MTS (3Å)] or iodine was used, the final concentrations were 0.05 mM and 2.5 μM, respectively. In most cases, the reactions were terminated by addition of 5 mM N-ethylmaleimide. While studying cross-linking rates, reactions were terminated at given times by addition of 5 mM methyl methanethiosulfonate and incubation on ice. Samples were mixed with loading buffer without DTT or heating and immediately subjected to NaDodSO4/10% PAGE, transferred onto poly(vinylidene difluoride) membranes (Immobilon-PVDF; Millipore) and immunoblotted with site-directed polyclonal antibody against the C terminus of LacY (residues 402–417; ref. 26).

Results

Intermolecular Cross-Linking.

Intermolecular cross-linking was tested with 250 LacY mutants containing single-Cys replacements in each transmembrane domain and in some helix-loop interfaces (Fig. 1). Almost the entire transmembrane region was scanned, including positions 14–32 (helix I), 47–68 (helix II), 74–91 (helix III), 113–133 (helix IV), 138–160 (helix V), 170–190 (helix VI), 222–247 (helix VII), 261–278 (helix VIII), 291–308 (helix IX), 315–335 (helix X), 346–370 (helix XI), and 382–402 (helix XII). Four single-Cys mutants expressed poorly, as reported previously [positions 296 (15), 358 (27), as well as 386 and 400 (21)], and were not tested.

Intact LacY electrophoreses with a molecular mass of about 32 kDa, and the LacY/LacY tandem fusion protein migrates at about 58 kDa (6). Mutants with a single Cys residue at position 78 (helix III); positions 185, 186, and 187 (helix VI); positions 275, 278, and 263 (helix VIII); or position 308 (helix IX) exhibit a band corresponding to the LacY/LacY tandem fusion protein after treatment with the cross-linking reagent MTS-3-MTS (0.05 mM; 5 Å; Fig. 2A). The mutant with a single-Cys at position 398 (helix XII) contains a biotin acceptor domain (BAD) in loop VI-VII, and therefore exhibits a band corresponding to the molecular mass of LacY/LacY with the BAD (80 kDa) after cross-linking (Fig. 2B). In contrast, control samples treated with 2.5% dimethyl sulfoxide (DMSO), the solvent for the cross-linking agent, do not exhibit such a band. Thus, these positions are probably located on exposed surfaces of LacY so that intermolecular cross-links are formed producing homodimers. Of 250 single-Cys mutants tested, only these 9 mutants exhibit significant intermolecular cross-linking under the conditions described.

Figure 2.

Figure 2

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Intermolecular thiol cross-linking of LacY mutants with single Cys replacement at given positions. Membranes prepared from E. coli T184 containing single-Cys LacY mutants were cross-linked with 0.05 mM MTS-3-MTS (5 Å) at room temperature for 1 h, and reactions were terminated with 5 mM N-ethylmaleimide as described in Experimental Procedures. NaDodSO4/10% PAGE and immunoblotting were also performed as described. (A) LacY monomer electrophoreses with a molecular mass of about 32 kDa, and LacY/LacY tandem fusion dimer migrates with a molecular mass of about 58 kDa. Mutants that form homodimers after treatment with MTS-3-MTS (+) migrate about 58 kDa, the same molecular mass as observed with LacY/LacY tandem dimer. Control samples (−) were treated with 2.5% DMSO, the solvent for the cross-linking agent. (B) Mutant F398C contains a biotin acceptor domain in loop VI-VII and has a molecular mass of about 46 kDa; the cross-linked dimer migrates with a molecular mass of about 80 kDa.

Three single-Cys mutants at positions 275, 278 (helix VIII) or 398 (helix XII) were studied further with the shorter cross-linking reagent MTS-1-MTS (3 Å). The mutants exhibit similar cross-linking efficiency with this reagent (Fig. 3 A and B). However, no significant homodimer formation is observed with iodine as an oxidizing agent (data not shown). Furthermore, when cross-linking is carried out at 0°C, efficiency is markedly decreased (Fig. 4A, lanes 3 and 7; Fig. 4B, lane 3). This phenomenon is also observed with mutant F185C and F186C, although the data are not shown. Finally, the high-affinity substrate analog β,D-galactopyranosyl 1-thio-β,D-galactopyranoside has no significant effect on cross-linking with single-Cys residues at positions 275 and 278 (helix VIII); 398 (helix XII; Fig. 4); or positions 185, 186, or 187 in helix VI (data not shown).

Figure 3.

Figure 3

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Cross-linking with MTS-1-MTS vs. MTS-3-MTS. Cross-linking was carried out with 0.05 mM cross-linking reagents at room temperature for 1 h, and the samples were treated as described in Fig. 2. (A) Mutants I275C and F278C; (B) mutant F398C with a biotin acceptor domain in loop VI-VII.

Figure 4.

Figure 4

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Effect of incubation at 0°C or ligand on cross-linking. As indicated, intermolecular cross-linking was carried out either at 0°C or in the presence of 10 mM β,D-galactopyranosyl 1-thio-β,D-galactopyranoside (TDG) at room temperature (RT) for 1 h. Control lanes represent samples incubated with 2.5% DMSO alone. (A) Mutants I275C and F278C; (B) mutant F398C with a biotin acceptor domain in loop VI-VII.

Cys Residues on Opposite Sides of LacY Cross-Link to Form Homodimers.

The rate of cross-linking with two mutants containing single Cys residues on opposite faces of 12-helix bundle that comprises LacY (positions 278 in helix VIII and 398 in helix XII) is shown in Fig. 5 A and B. Cys mutant 278 cross-links somewhat more efficiently than Cys mutant 398, but both mutants exhibit similar time courses of cross-linking, and in both instances, the marked sigmoidicity is observed (Fig. 5C). Thus, the rate of cross-linking increases slowly over the initial 3 min, abruptly between 3 and 5 min and at a slower rate thereafter. Although data are not shown, each of the other mutants that exhibit homodimer formation under these conditions also exhibit sigmoid time courses.

Figure 5.

Figure 5

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Rate of cross-linking with mutant F278C or F398C with a biotin acceptor domain in loop VI-VII. Cross-linking was carried out with 0.05 mM MTS-3-MTS at room temperature. Reactions were terminated at given times by addition of 5 mM methyl methanethiosulfonate (MMTS) and placed on ice. (A) mutant F278C; (B) mutant F398C with a biotin acceptor domain in loop VI-VII; (C) cross-linking as a function of time. Bands corresponding to given uncross-linked and cross-linked fragments were quantified with a ChemiImager (Alpha Innotech, San Leandro, CA). The ordinate represents the intensity of the band corresponding to the homodimer relative to the combined intensities of the uncross-linked and cross-linked bands expressed as a percentage.

Discussion

Intermolecular thiol cross-linking was used to scan 250 LacY mutants with single-Cys replacements located in the transmembrane domains and in some helix-loop boundaries. Only 9 mutants [W78C (helix III); F185C, F186C, and A187C (helix VI); Y263C, I275C, and F278C (helix VIII); F308C (helix IX); and F398C (helix XII)] exhibit intermolecular cross-linking by using a homobifunctional MTS cross-linking agent that is 5 Å in length (Figs. 1 and 2). The results are consistent with the LacY helix packing model in the sense that all 9 positions are located on the periphery of the 12-helix bundle and face away from the middle of the molecule (Fig.6). It is also noteworthy that all 9 mutants are at or near helix-loop interfaces and that the native residue in each instance is either hydrophobic or contains an aromatic ring (Fig. 1). Finally, 7 of the 9 mutants are located toward the cytoplasmic surface, whereas only 2 are located toward the periplasmic surface, suggesting that the profile of LacY in the membrane may not be regular (28–30).

The number of single-Cys mutants that exhibit intermolecular cross-linking is surprisingly small. An irregularly shaped molecule may account in part for the small number of mutants that cross-link. However, in view of the observation that all of the productive positions are located at the ends of helices or at helix-loop boundaries, it is also likely that Cys residues facing the interior of the bilayer are relatively unreactive. In the low dielectric of the bilayer, the thiol form is greatly favored over the thiolate anion, and the latter is far more reactive (31). In addition, most homobifunctional cross-linking agents, more specifically the bis-MTS reagents used here, are amphipathic, which may limit accessibility to Cys residues buried in the bilayer. Although relatively few mutants cross-link to form homodimers, many of the single-Cys mutants tested react with N-ethylmaleimide and/or thiosulfonate derivatives (13, 32–35). Therefore, another possibility for the low abundance of homodimers is that one functional group in the cross-linking agent reacts with Cys replacements facing the interior of the molecule so that the other reactive group is never in sufficiently close proximity to react with the homolous Cys residue in another molecule. Finally, the position that defines the ends of the transmembrane helices is an approximation based on single amino acid deletion analysis (36, 37) that is probably accurate to within one turn of a helix. Because it appears that only residues at helix-loop interfaces undergo cross-linking, another possibility for the small number of single-Cys mutants that cross-link is that the ends of some of the transmembrane helices have been underestimated, leading to artifactually negative observations for some of the positions tested.

Although it has been suggested that LacY may function as a dimer under certain conditions (38–40), more direct observations (6, 29, 41–43) provide convincing evidence that LacY is both structurally and functionally a monomer. Data presented in this study strongly support this contention. (i) In addition to their location at the end of helices or at helix-loop boundaries, the 9 Cys-replacement mutants that exhibit homodimer formation are all positioned on the periphery of the 12-helix bundle facing away from the middle of LacY (Fig. 6). (ii) Mutants F275C and F398C located on opposite sides of the 12-helix bundle cross-link at rates within the same order of magnitude. (iii) The time course of homodimer formation with mutants F275C or F398C is markedly sigmoid. This behavior is consistent with the interpretation that, in order for homodimer formation to occur, two separate reactions that occur at different rates are required. First, there is a relatively rapid reaction in which one functional group in the cross-linking agent reacts with a Cys residue. In a second, much slower reaction, the second functional group reacts with the Cys in the second molecule to produce a homodimer. Clearly, the second reaction must be much slower than the first, as it requires that appropriate faces of two LacY molecules collide within the plane of the membrane. (iv) Homodimer formation is markedly inhibited at 0°C. Although inhibition may be due in part to the effect of temperature on chemical reactivity per se, it is well known that the rate of lateral diffusion is dramatically decreased below the lipid phase transition (reviewed in ref. 44), which occurs at 18°C in E. coli (45). (v) Intramolecular cross-linking in functional split LacY constructs (see ref. 46; L.G. and H.R.K., unpublished observations) exhibit little or no sigmoidicity. (vi) Finally, it is noteworthy that single-Cys mutants at position 101 (periplasmic loop III-IV), position 313 (periplasmic loop IX/X), or position 375 (periplasmic loop XI/XII) cross-link spontaneously under atmospheric conditions (47). Taken as a whole, the observations provide strong support for the interpretation that LacY is both structurally and functionally a monomer and that homodimer formation in certain single-Cys mutants occurs as the result of a stochastic process involving random collisions between monomers.

Acknowledgments

We thank Miklós Sahin-Tóth for many useful discussions and valuable suggestions and Yonglin Hu for doing the graphics for Fig. 6. We are also indebted to Gregory Kaczorowski and Nancy Carrasco for reading the manuscript and making editorial suggestions. This work was supported in part by National Institutes of Health Grant DK51131:06 to H.R.K.

Abbreviations

LacY

lactose permease

MTS-1-MTS

1,1-methanediyl bismethanethiosulfonate

MTS-3-MTS

1,3-propanediyl bismethanethiosulfonate

C6

the six C-terminal transmembrane helices

BAD

biotin acceptor domain

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