The C-4 Hydroxyl Group of Galactopyranosides Is the Major Determinant for Ligand Recognition by the Lactose Permease of <i>Escherichia coli</i>

Sahin–Tóth, Miklós; Mc, Lawrence; Nishio, Takeshi; Hr, Kaback

doi:10.1021/bi011233l

Cited by 44 publications

(74 citation statements)

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“…The galactopyranosyl ring, which contains all the determinants for specificity, is shown with an MTS group at the anomeric position. The C-4 OH is most important with respect to specificity (18). Cys 148 (helix V) interacts weakly and hydrophobically with the galactopyranosyl ring, and position 122 (helix IV; shown as Ala) is in close proximity to the reactive thiol of the MTS moiety (24).…”

Section: Discussionmentioning

confidence: 99%

See 1 more Smart Citation

Probing the Mechanism of a Membrane Transport Protein with Affinity Inactivators

Guan

Sahin–Tóth

Kálai

et al. 2003

Journal of Biological Chemistry

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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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Section: Discussionmentioning

confidence: 99%

“…The specificity of LacY is directed toward the galactosyl moiety of the substrate, and the C-4 OH is most important for ligand binding (16,18). The major determinants for substrate binding are located at the interface between helices IV, V (19 -24), and VIII (50).…”

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confidence: 99%

Probing the Mechanism of a Membrane Transport Protein with Affinity Inactivators

Guan

Sahin–Tóth

Kálai

et al. 2003

Journal of Biological Chemistry

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“…Because the C-4 OH group is clearly the most important determinant for recognition (17,19) and Arg-144 seems to be too distant to H bond with this position, a likely candidate for the role is Glu-269, which is located at the interface between helix VIII and helix V (14,38,39). Furthermore, evidence has been presented (21,(40)(41)(42) indicating that this residue is critical for substrate recognition.…”

Section: Discussionmentioning

confidence: 99%

“…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).…”

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confidence: 99%

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

Guan

Sahin–Tóth

Kaback

2002

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

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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 ...

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“…10 Moreover, 4-and 3-hydroxyl are important in galactoside recognition of LacY 11 and the order of importance to binding affinity is 4-hydroxyl >> 6-hydroxyl > 3-hydroxyl. 12 The protein specificity is independent of the anomeric state of the sugar, but the affinity of α-anomeric states of disaccharides is greater than β. 11,13 The atomic-level description for the cause of the anomeric affinity is unknown.…”

Section: Introductionmentioning

confidence: 99%

Sugar Binding in Lactose Permease: Anomeric State of a Disaccharide Influences Binding Structure

Klauda

Brooks

2007

Journal of Molecular Biology

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Lactose permease in E. coli (LacY) transports both anomeric states of disaccharides but has greater affinity for α-sugars. Molecular dynamics (MD) simulations are used to probe the protein-sugar interactions, binding structures, and global protein motions in response to sugar binding by investigating LacY (the experimental mutant and wild-type) embedded in a fully hydrated lipid bilayer. A total of twelve MD simulations of 20-25ns each with β(α)-D-galactopyranosyl-(1,1)-β-Dgalactopyranoside (ββ-(Galp) 2 ) and αβ-(Galp) 2 result in binding conformational families that depend on the anomeric state of the sugar. Both sugars strongly interact with Glu-126 and αβ-(Galp) 2 has a greater affinity to this residue. Binding conformations are also seen that involve protein residues not observed in the crystal structure, as well as those involved in the proton translocation (Phe-118, Asn-119, Asn-240, His-322, Glu-325, and Tyr-350). Common to nearly all protein-sugar structures, water acts as a hydrogen bond bridge between the disaccharide and protein. The average binding energy is more attractive for αβ-(Galp) 2 than ββ-(Galp) 2 , i.e., −10.7±0.7 and −3.1±1.0 kcal/mol, respectively. Of the twelve helices in LacY, Helix-IV is the least stable with ββ-(Galp) 2 binding resulting in larger distortion than αβ-(Galp) 2 .

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The C-4 Hydroxyl Group of Galactopyranosides Is the Major Determinant for Ligand Recognition by the Lactose Permease of Escherichia coli

Cited by 44 publications

References 30 publications

Probing the Mechanism of a Membrane Transport Protein with Affinity Inactivators

Probing the Mechanism of a Membrane Transport Protein with Affinity Inactivators

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

Sugar Binding in Lactose Permease: Anomeric State of a Disaccharide Influences Binding Structure

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