Molecular biology of the lactose carrier of Escherichia coli

Journal of Biological Chemistry

Sahin–Tóth

Kálai

et al. 2003

Affinity inactivators are useful for probing catalytic mechanisms. Here we describe the synthesis and properties of methanethiosulfonyl (MTS) galactose or glucose derivatives with respect to a well studied membrane transport protein, the lactose permease of Escherichia coli. The MTS-galactose derivatives behave as affinity inactivators of a functional mutant with Ala 122 3 Cys in a background otherwise devoid of Cys residues. A proton electrochemical gradient (⌬ H ؉) markedly increases the rate of reaction between Cys 122 and MTS-galactose derivatives; nonspecific labeling with the corresponding MTS-glucose derivatives is unaffected. When the Ala 122 3 Cys mutation is combined with a mutation (Cys 154 3 Gly) that blocks transport but increases binding affinity, discrimination between the MTS-galactose and -glucose derivatives is abolished, and ⌬ H ؉ has no effect. The results provide strong confirmation that the non-galactosyl moiety of permease substrates abuts Ala 122 in helix IV. In addition, the findings demonstrate that the MTS-galactose derivatives do not react with the Cys residue at position 122 upon binding per se but at a subsequent step in the overall transport mechanism. Thus, these inactivators behave as unique suicide substrates.The lactose permease (LacY) 1 of Escherichia coli transduces the free energy stored in an electrochemical H ϩ gradient into a concentration gradient of D-galactopyranosides and vice versa (galactoside/H ϩ symport) (1-4). LacY is a 12-transmembranehelix bundle with the N and C termini on the cytoplasmic face of the membrane (5-7). Several lines of evidence indicate that it is both functionally (8) and structurally a monomer (9 -11).Analysis of an extensive library of mutants, particularly Cys replacement mutants (12), with a battery of site-directed biophysical and biochemical techniques has led to the formulation of a teriary structure model (49) that includes tilts as well as a working model for the transport mechanism (13).LacY is in close proximity to the nongalactosyl portion of ligand (24). Affinity labeling is based upon specific recognition of an inactivating ligand at the binding site. Because Cys 148 (helix V), which is in close proximity to Ala 122 , makes direct contact with the galactopyranosyl moiety of the substrate, numerous attempts to develop an affinity reagent specific for this side chain have failed. On the other hand, Ala 122 abuts the nongalactosyl moiety of disaccharide substrates, and alkyation of a Cys residue at this position blocks binding of disaccharides with no effect on galactose transport or binding. Therefore, methanethiosulfonyl (MTS) galactoside with an ethylene linkage between the galactopyranoside ring and MTS and MTS-5-galactose with a carbamide linkage were synthesized as potential affinity reagents for the single-Cys A122C mutant in LacY with the corresponding glucosides as controls. EXPERIMENTAL PROCEDURES ReagentsMTS-galactoside and MTS-glucoside-MTS-galactoside and MTSglucoside were synthesized as shown in the synthetic scheme (Sch...

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confidence: 99%

Probing the Mechanism of a Membrane Transport Protein with Affinity Inactivators

Journal of Biological Chemistry

Sahin–Tóth

Kálai

et al. 2003

“…[1][2][3][4]. LacY is a 12-transmembrane-helix bundle with the N and C termini on the cytoplasmic face of the membrane (5)(6)(7), and the 417-residue polypeptide has been solubilized, purified, reconstituted into proteoliposomes, and shown to be solely responsible for galactoside͞H ϩ symport (reviewed in ref.…”

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confidence: 99%

Changing the lactose permease of Escherichia coli into a galactose-specific symporter

Proc. Natl. Acad. Sci. U.S.A.

Sahin–Tóth

Kaback

2002

T he lactose permease (LacY) (1) of Escherichia coli is a paradigm for a major class of membrane transport proteins that transduce free energy stored in electrochemical ion gradients into solute concentration gradients (reviewed in refs. 1-4). LacY is a 12-transmembrane-helix bundle with the N and C termini on the cytoplasmic face of the membrane (5-7), and the 417-residue polypeptide has been solubilized, purified, reconstituted into proteoliposomes, and shown to be solely responsible for galactoside͞H ϩ symport (reviewed in ref. 8). Several lines of evidence indicate that LacY is both functionally (9) and structurally a monomer (10-12). Analysis of an extensive library of mutants, particularly Cys replacement mutants (13), with a battery of site-directed biophysical and biochemical techniques has led to the formulation of a helix-packing model including tilts, as well as the working model for the mechanism of LacY (reviewed in ref. 14).LacY is selective for disaccharides containing a Dgalactopyranosyl ring, as well as D-galactose (15-17), but has no affinity for D-glucopyranosides or D-glucose (17, 18). The specificity of LacY is directed toward the galactosyl moiety of the substrate, and although the C-4 OH is clearly the major determinant for ligand binding, the C-2, C-3, and C-6 OH groups also participate in H-bonding interactions (17, 19).Glu-126 and Arg-144, which are charge-paired, are irreplaceable determinants for substrate recognition and located at the interface between helices IV and V, respectively (20-23). Although Cys-148 (helix V) is not irreplaceable, it was the first residue shown to interact directly with substrate (18,(24)(25)(26)(27)(28). Alkylation with N-ethylmaleimide (NEM) abolishes lactose transport and p-nitrophenyl ␣,D-galactopyranoside binding, and substrates that include D-galactose and D-galactopyranosides afford protection against reaction with NEM and other thiol reagents (see ref. 14). Furthermore, replacement of Cys-148 with small hydrophobic residues (e.g., Ala, Val) increases apparent affinity for lactose (i.e., K m decreases), whereas hydrophilic replacements (e.g., Ser, Thr) increase K m . In addition, hydrophilic replacements decrease transport of D-galactose relative to galactopyranosides (26). The results indicate that Cys-148 interacts with the hydrophobic face of the galactopyranosyl moiety of the disaccharides or galactose.Site-directed NEM labeling of single-Cys mutants in helices IV and V (29) reveals that mutant A122C (helix IV) exhibits properties similar to those of native Cys-148. Thus, NEM inactivates lactose transport and ligand affords protection against inactivation, as well as alkylation by NEM. Moreover, Ala-122 is located at about the same level in the membrane as Cys-148 (23). Therefore, it was suggested that Ala-122 may be a component of the substrate-binding site.In this study, we determined the effect of site-directed chemical modification of single-Cys A122C LacY and site-directed mutagenesis of Ala-122 on transport activity and͞or substratebinding ...

“…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)(2)(3)(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.…”

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confidence: 99%

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

Proc. Natl. Acad. Sci. U.S.A.

Murphy

Kaback

2002

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...