Probing the Mechanism of a Membrane Transport Protein with Affinity Inactivators*

Lan GuanDagger , Miklós Sahin-TóthDagger , Tamás Kálai§, Kálmán Hideg§, and H. Ronald KabackDagger

From the Dagger  Howard Hughes Medical Institute, Departments of Physiology and Microbiology & Molecular Genetics and the Molecular Biology Institute, UCLA, Los Angeles, California 90095-1662 and the § Institute of Organic and Medicinal Chemistry, University of Pécs, H-7643 Pécs, Hungary

Received for publication, November 6, 2002, and in revised form, December 4, 2002

    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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 Ala122right-arrowCys in a background otherwise devoid of Cys residues. A proton electrochemical gradient (Delta <A><AC>&mgr;</AC><AC>&cjs1171;</AC></A>H+) markedly increases the rate of reaction between Cys122 and MTS-galactose derivatives; nonspecific labeling with the corresponding MTS-glucose derivatives is unaffected. When the Ala122right-arrowCys mutation is combined with a mutation (Cys154right-arrowGly) that blocks transport but increases binding affinity, discrimination between the MTS-galactose and -glucose derivatives is abolished, and Delta <A><AC>&mgr;</AC><AC>&cjs1171;</AC></A>H+ has no effect. The results provide strong confirmation that the non-galactosyl moiety of permease substrates abuts Ala122 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.

    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

The lactose permease (LacY)1 of Escherichia coli transduces the free energy stored in an electrochemical H+ gradient into a concentration gradient of D-galactopyranosides and vice versa (galactoside/H+ symport) (1-4). LacY is a 12-transmembrane-helix 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 selective for disaccharides containing a D-galactopyranosyl ring as well as D-galactose (14-16), but it has no affinity for D-glucose or D-glucopyranosides (16, 17). 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). Alkylation of single-Cys122 LacY (helix IV) with N-ethylmaleimide (NEM) abolishes lactose transport as well as ligand binding. Moreover, lactose, melibiose, and beta -D-galactopyranosyl 1-thio-beta -D-galactopyranoside (TDG) protect against NEM inactivation of lactose transport as well as labeling of Cys122 by [14C]NEM. Remarkably, however, D-galactose transport is essentially unaffected by NEM, and the monosaccharide affords no protection against inactivation of lactose transport or labeling of Cys122 by NEM. Consistently, competitive inhibition of galactose transport by lactose, melibiose, or TDG is drastically reduced after alkylation by NEM, whereas inhibition by unlabeled galactose is unaffected. Finally, replacement of Ala122 with Phe or Tyr has the same effect as alkylation of single-Cys mutant A122C with NEM. The results demonstrate that Ala122 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 Cys148 (helix V), which is in close proximity to Ala122, 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, Ala122 abuts the non-galactosyl 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
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Reagents

MTS-galactoside and MTS-glucoside---MTS-galactoside and MTS-glucoside were synthesized as shown in the synthetic scheme (Scheme 1). Reagents and conditions were as follows: (i) bromoethanol 1.25 eq, BF3Et2O 5.1 eq, CH2Cl2, 0 °C right-arrow room temperature, 24 h, (53-61%); (ii) NaOMe 0.1 eq, MeOH, 4 h, and then Dowex 50-W (H+), NaSSO2CH3 (1.3 eq), N,N-dimethylformamide, 50 °C, 16 h (36-42%).


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Scheme 1.   Synthetic scheme.

Melting points were determined with a Boetius micro melting point apparatus and are uncorrected. A synthesis of compounds 3a and 3b was published previously (25). All of the chemical and physical data were identical to those published previously. All of the chemicals were purchased from Sigma, and the NaSSO2CH3 was synthesized as described by Mintel and Westley (26). Preparative flash column chromatography was performed on Merck Kieselgel 60 (0.040-0.063 mm). Qualitative TLC was carried out on commercially prepared plates (20 DC-Alufolien 5 × 7.5 cm) coated with Merck Kieselgel 60 F254 and visualization was achieved by heating the developed plate.

2-(2,3,4,6-Tetra-O-acetyl-beta -D-galactopyranosyl)ethyl Bromide (2a) and 2-(2,3,4,6-Tetra-O-acetyl-beta -D-glucopyranosyl)ethyl Bromide (2b)-- To a stirred solution of beta -D-galactose-pentaacetate (1a) or beta -D-glucose-pentaacetate (1b) (3.9 g, 10.0 mmol) and bromoethanol (0.88 ml, 12.4 mmol), BF3·Et2O (6.5 ml, 51.0 mmol) in CH2Cl2 (20 ml) was added dropwise at 0 °C, and the mixture was allowed to warm to room temperature and stirred for an additional 24 h. The mixture was poured over ice-water (20 ml) and separated, and the aqueous phase was washed with CH2Cl2 (3 × 10 ml); the combined organic phase was washed with 5% aqueous NaHCO3 (10 ml) and water (10 ml), dried (MgSO4), filtered, and evaporated; and the residue was purified by flash column chromatography (hexane/EtOAc) to give the title compounds 2a (2.77 g (61%), melting point 108-110 °C, Rf 0.35 (hexane/EtOAc, 2:1)) and 2b (2.41 g (53%) 113-115 °C, Rf 0.32 (hexane/EtOAc, 2:1)).

2-(beta -D-Galactopyranosyl)ethyl Methanethiosulfonate (3a) and 2-(beta -D-Glucopyranosyl)ethyl Methanethiosulfonate (3b)-- To a stirred solution of compound 2a or 2b (455 mg, 1.0 mmol) in MeOH (10 ml), a freshly made 0.1 M NaOMe solution in MeOH (1 ml) was added, and the mixture was stirred for further 4 h. The reaction mixture was then passed through a Dowex 50-W (H+) (2.5 × 2-cm plug, eluant MeOH), and the solvent was removed in vacuo to give an oil 215 mg (74%) for 3a and 200 mg (69%) for 3b. This product was immediately dissolved in N,N-dimethylformamide (10 ml); NaSSO2CH3 (134 mg, 1.0 mmol) was added, and the mixture was heated at 50 °C for 15 h. Then, N,N-dimethylformamide was evaporated, and the residue was purified by flash column chromatography (EtOAc/MeOH) to give the title compounds as white thick oils that solidified upon cooling (3a, 133 mg (42%), Rf 0.38 (EtOAc/MeOH, 4:1); 3b, 114 mg (36%), Rf 0.48 (EtOAc/MeOH, 4:1)).

MTS-5-galactose and -glucose---MTS-5-galactose was custom synthesized by Toronto Research Chemicals (Toronto, Canada), and MTS-5-glucose was purchased from the same source.

Other Reagents-- [1-14C]Lactose was obtained from Amersham Biosciences. N-([14C]ethyl)Maleimide was purchased from PerkinElmer Life Sciences. [3H]NPG was the generous gift of Gérard Leblanc (Villefranche-sur-mer, France). Immobilized monomeric avidin was from Pierce, and all unlabeled sugars were obtained from Sigma. All other materials were reagent grade and were obtained from commercial sources.

Construction of LacY Mutants

All mutants were constructed in a cassette lacY gene encoding functional LacY devoid of Cys residues with a C-terminal biotin acceptor domain (27, 28). Plasmids pKR35/A122C/C148S and pKR35/A122C/C148A have been described (24, 29). pKR35/A122C/C148D and pKR35/A122C/C148A/C154G were generated by oligonucleotide-mediated, site-directed mutagenesis using two-step PCR with pKR35/A122C/C148A as template.

Growth of Cells

E. coli T184 (lacI+O+Z-Y-(A) rpsL,met-,thr-,recA,hsdM,hsdR/F',lacIqO+ZD118(Y+A+)) containing given mutants was grown in Luria-Bertani broth with 100 mg/liter of ampicillin. Overnight cultures were diluted 10-fold and allowed to grow for 2 h at 37 °C before induction with 1 mM isopropyl 1-thio-beta -D-galactopyranoside. After additional growth for 2-3 h at 37 °C, cells were harvested by centrifugation.

Preparation of Right-Side-Out Vesicles

Right-side-out (RSO) membrane vesicles were prepared by osmotic lysis as described (30, 31), suspended in 100 mM potassium Pi (pH 7.5)/10 mM MgSO4 at a protein concentration of about 12 mg/ml, frozen in liquid N2, and stored at -80 °C until use.

Inactivation by MTS Sugars

RSO vesicles at a protein concentration of 3 mg/ml were incubated at 24 °C with a given MTS sugar in the absence or presence of 20 mM ascorbate and 0.2 mM phenazine methosulfate (PMS) under oxygen (32, 33). Reactions (0.25 ml, total volume) were quenched by the addition of 1.75 ml of ice-cold 100 mM potassium Pi (pH 7.5)/10 mM MgSO4 at the indicated times and immediately centrifuged. The pellet was washed with 2.0 ml of the same buffer, centrifuged, and resuspended in 0.25 ml of 100 mM potassium Pi (pH 7.5)/10 mM MgSO4.

Transport Assays

Lactose transport with [1-14C]lactose (10 mCi/mmol) at a final concentration of 0.4 mM was carried out in the presence of 20 mM ascorbate and 0.2 mM PMS under oxygen as described previously (33). For mutant A122C/C148D, measurements of the rate of lactose transport were studied with vesicles washed in 100 mM potassium Pi/10 mM MgSO4 (pH 5.5), because this mutant exhibits a higher rate of transport at acidic pH (34).

NEM Labeling

The Kd for TDG binding was determined in situ by alkylation of given mutants with [14C]NEM in the absence or presence of given concentrations of TDG (35, 36).

To determine the reaction of Cys122 with MTS sugars, vesicles were incubated with an MTS sugar at a given concentration for indicated periods of time, diluted and washed with 100 mM potassium Pi (pH 7.5)/10 mM MgSO4 as described above, and concentrated to a protein concentration of 15 mg/ml. [14C]NEM (40 mCi/mmol; 0.5 mM final concentration) was added for 30 min, and the reaction was quenched by adding 20 mM dithiothreitol. The samples were then treated as described (29, 35).

Flow Dialysis

Binding of [3H]NPG to RSO vesicles containing given LacY mutants was measured by flow dialysis as described (37).

Determination of Methanthiosulfonate Concentration

The concentration of MTS reagents was determined by measuring the disulfide exchange among 5,5-dithio-bis-(2-nitrobenzoic acid), dithiothreitol, and MTS (38).

    RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

MTS Sugars-- MTS-galactoside and MTS-glucoside (Fig. 1) were synthesized as described under "Experimental Procedures." MTS-5-galactose and MTS-5-glucose were custom-synthesized by Toronto Research Chemicals.


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Fig. 1.   Structure of MTS sugars. Note the ethylene linkage bridging the pyranosyl rings of MTS-galactoside and -glucoside and the carbamide linkage in MTS-5-galactose and -glucose, as well as the increased length of the latter pair.

Inactivation of LacY by MTS Sugars-- When RSO membrane vesicles expressing single-Cys mutant A122C with Cys148 replaced by Ala were treated with given concentrations of MTS-galactoside or MTS-glucoside for 10 min and initial rates of lactose transport were measured, EC50 values (the concentration at which half-maximal inhibition is observed) approximating 36 and 130 µM, respectively, were obtained (Fig. 2A, Table I). However, it is noteworthy that at low concentrations of MTS-galactoside, a significant percentage of the reagent reacts with Cys residues unassociated with LacY, making it difficult to determine a true EC50.2 MTS-5-galactose, which is more hydrophilic because of the carbamide linkage (Fig. 1), exhibits an EC50 of about 151 µM, whereas MTS-5-glucose has an EC50 well in excess of 800 µM (Fig. 2B).


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Fig. 2.   Concentration-dependent inactivation of lactose transport and blockade of NEM labeling of A122C/C148A by MTS sugars. RSO vesicles containing mutant A122C/C148A were incubated with given concentrations of MTS sugars for 10 min. Samples were washed and assayed for lactose transport or labeled with [14C]NEM as described under "Experimental Procedures." Initial rates of transport were measured over a time course of 0 to 15 s and expressed as a percentage of the rate obtained in the absence of inhibitor. A, inactivation of lactose transport by MTS-galactoside and MTS-glucoside; B, inactivation of lactose transport by MTS-5-galactose and MTS-5- glucose; C, blockade of [14C]NEM labeling by MTS-galactoside or MTS-glucoside.


                              
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Table I
Inactivation by MTS sugars
Kd values of given single-Cys122 mutants for TDG were measured by protection against [14C]NEM labeling as described under "Experimental Procedures." Initial rates of lactose transport were measured over a time course of 0, 5, 10, and 15 s (Figs. 3A and 6B). EC50 values for MTS-galactoside and MTS-glucoside were determined as described in Fig. 2A. Rates of reaction were determined as described in Fig. 3, B and C, and pseudo-first-order rate constants (b) were obtained from a nonlinear least-squares fit of the data to equation a × exp(-bx) + c. The second-order rate constants (k) were calculated by dividing b by the concentration of the inactivator. Values given for A122C/C148A/C154G were obtained from experiments in which the blockade of [14C]NEM labeling by MTS-galactoside or glucoside was determined. ND, not determined.

To demonstrate that the differential effect of MTS-galactoside and MTS-glucoside on transport is due specifically to the reaction with the Cys residue at position 122, blockade of [14C]NEM labeling by the MTS sugars was studied (Fig. 2C). RSO vesicles were treated under the same conditions as described for transport and then labeled with NEM. Prior to treatment with MTS-galactoside, Cys122 reacted well with NEM, and treatment with MTS-galactoside at concentrations ranging from 50 to 400 µM blocked reactivity completely. In contrast, at 50 µM MTS-glucoside, little or no effect on NEM labeling was observed, 100 µM blocked NEM labeling by about 50%, and complete abrogation was observed at 400 µM. Thus, there is good correlation between inhibition of transport activity and reactivity with the Cys residue at position 122. Consistent with this interpretation, under the same conditions, no inactivation whatever was observed with functional LacY devoid of Cys residues (data not shown).

If MTS-galactoside is an affinity reagent for the A122C/C148A mutant, altering the affinity of LacY for substrate should be reflected in the time course of MTS-galactoside inactivation. As shown in Fig. 3A and Table I, mutants containing a single Cys residue at position 122 and Ala, Ser, or Asp at position 148 exhibit decreasing initial rates of lactose transport. Moreover, TDG protection against NEM labeling of Cys122 (24, 35) reveals Kd values of 0.2, 3, and 40 mM, respectively (Table I). Although there is no quantitative relationship between the initial rate and Kd, qualitatively, A122C/C148A LacY exhibited the best affinity and the highest rate of transport followed by mutants C148S and C148D (Fig. 3A; Table I). Relative to mutant A122C/C148A, which exhibits a second-order rate constant (k) of ~235 M-1 s-1 for MTS-galactoside inactivation, mutants A122C/C148S and A122C/C148D exhibit rate constants of about 56 and 31 M-1 s-1, respectively (Fig. 3B; Table I), indicating that the reaction rates correlate qualitatively with binding affinity and transport activity. In contrast, inactivation by MTS-glucoside exhibits essentially the same rate of inactivation of lactose transport by the three mutants and a second-order rate constant that averages 11 M-1 s-1 (Fig. 3C; Table I). Thus, MTS-galactoside inactivates mutant A122C/C148A a minimum of 21 times faster than MTS-glucoside. Taken together, the results indicate that MTS-galactoside is an affinity reagent. In contrast, MTS-glucoside reacts nonspecifically like many other thiol-reactive reagents.


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Fig. 3.   Time course of inactivation of lactose transport by MTS sugars in single Cys122 mutants with different sugar binding affinities. A, lactose transport in RSO vesicles containing mutants A122C/C148A, A122C/C148S, and A122C/C148D. B and C, rates of inactivation with MTS-galactoside or MTS-glucoside in given single Cys122 mutants. Vesicles were treated with 80 µM MTS-galactoside or 300 µM MTS-glucoside, stopped at given times by dilution, washed, and assayed for lactose transport as described under "Experimental Procedures." Rates of transport are expressed as a percentage of the rate obtained in the absence of inhibitors. black-square, A122C/C148A; , A122C/C148S; black-triangle, A122C/C148D.

Delta <A><AC>&mgr;</AC><AC>&cjs1171;</AC></A>H+ Increases Reaction Rates-- Because it is possible that MTS-galactoside and MTS-5-galactose may be actively transported, the effect of Delta <A><AC>&mgr;</AC><AC>&cjs1171;</AC></A>H+ (interior negative) on the rate of inactivation of lactose transport was studied in the absence and presence of reduced PMS under oxygen (32, 33), conditions known to generate a large membrane potential at pH 7.5 (39-41). In the absence of Delta <A><AC>&mgr;</AC><AC>&cjs1171;</AC></A>H+, 100 µM MTS-galactoside inhibits relatively slowly, yielding a second-order rate constant (k) of 220 M-1 s-1 (Fig. 4A; Table II). Remarkably, in the presence of Delta <A><AC>&mgr;</AC><AC>&cjs1171;</AC></A>H+, at 100 µM MTS-galactoside, the rate of inactivation is too rapid to measure (data not shown). Although 20 µM MTS-galactoside does not inactivate in the absence of Delta <A><AC>&mgr;</AC><AC>&cjs1171;</AC></A>H+ (Fig. 2A), rapid inactivation is observed in the presence of Delta <A><AC>&mgr;</AC><AC>&cjs1171;</AC></A>H+ with a k = 10500 M-1 s-1 (Fig. 4A; Table II). However, inactivation does not go to completion but plateaus at 10 s because the concentration of the inactivator is depleted due to reaction with Cys residues unassociated with LacY.2 In any case, Delta <A><AC>&mgr;</AC><AC>&cjs1171;</AC></A>H+ increases the rate of inactivation by 48-fold (Table II). As shown in Fig. 4B, similar results are obtained with MTS-5-galactose; with this reagent, a 27-fold increase in the rate of inactivation is observed in the presence of Delta <A><AC>&mgr;</AC><AC>&cjs1171;</AC></A>H+ (Fig. 4B; Table II). With both inactivators, the effect of Delta <A><AC>&mgr;</AC><AC>&cjs1171;</AC></A>H+ is completely abolished in the presence of the protonophore carbonylcyanide-m-chlorophenylhydrazone, and neither inactivator has any effect whatever on the generation of Delta <A><AC>&mgr;</AC><AC>&cjs1171;</AC></A>H+, as judged by the accumulation of the lipophilic cation tetraphenylphosphonium (data not shown). In contrast, inactivation by MTS-glucoside is the same in the absence or presence of Delta <A><AC>&mgr;</AC><AC>&cjs1171;</AC></A>H+ over a wide range of concentrations (data not shown; see Fig. 5).


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Fig. 4.   Effect of Delta <A><AC>&mgr;</AC><AC>&cjs1171;</AC></A>H+ (interior negative) on the rate of inactivation by MTS sugars. RSO vesicles expressing A122C/C148A were treated with given concentrations of MTS-galactoside or MTS-glucoside, as indicated in the absence or presence of 20 mM ascorbate/0.2 mM PMS under oxygen, stopped at given times by dilution, washed, and assayed for lactose transport as described under "Experimental Procedures." Rates of transport are expressed as a percentage of the rate obtained in the absence of inactivators. A, MTS-galactoside; B, MTS-5-galactose.


                              
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Table II
Effect of Delta <A><AC>&mgr;</AC><AC>&cjs1171;</AC></A>H+ on the rate of inactivation of Cys122 by MTS sugars
RSO vesicles expressing A122C/C148A mutant were incubated in the absence or presence of given MTS sugars and 20 mM ascorbate/0.2 mM PMS under oxygen and then washed and treated as described under "Experimental Procedures." The second-order rate constants were determined from Fig. 4, A and B, according to the equation given in Table I.


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Fig. 5.   Effect of Delta <A><AC>&mgr;</AC><AC>&cjs1171;</AC></A>H+ on reaction of Cys122 with MTS sugars. RSO vesicles expressing mutant A122C/C148A were treated with given MTS sugars in the absence or presence of ascorbate and PMS under oxygen at given concentrations for 10 s, diluted, and washed. The vesicles were then concentrated and incubated with [14C]NEM as described under "Experimental Procedures."

The effect of Delta <A><AC>&mgr;</AC><AC>&cjs1171;</AC></A>H+ on the reactivity of the MTS sugars with Cys at position 122 was studied directly by blockade of NEM labeling (Fig. 5). In RSO vesicles with mutant A122C/C148A, Delta <A><AC>&mgr;</AC><AC>&cjs1171;</AC></A>H+ has no significant effect on the reactivity of Cys122 with NEM (Fig. 5, lanes 1 and 2). When treated with 100 µM MTS-galactoside or 400 µM MTS-5-galactose for 10 s in the absence of Delta <A><AC>&mgr;</AC><AC>&cjs1171;</AC></A>H+, no significant effect on NEM labeling was observed (compare Fig. 5, lanes 3 and 7 with lane 1). When the reaction was carried out after the generation of Delta <A><AC>&mgr;</AC><AC>&cjs1171;</AC></A>H+, NEM labeling was almost completely abolished (Fig. 5, lanes 4 and 8). In contradistinction, generation of Delta <A><AC>&mgr;</AC><AC>&cjs1171;</AC></A>H+ had no significant effect on NEM labeling with either of the glucose derivatives at the concentrations used (Fig. 5, lanes 5 and 6 and lanes 9 and 10). Thus, the marked increase in the rate of inactivation of transport with MTS-galactoside and MTS-5-galactose in the presence of Delta <A><AC>&mgr;</AC><AC>&cjs1171;</AC></A>H+ is due specifically to increased reaction with the Cys residue at position 122.

Inactivation by MTS-galactoside Occurs at a Step Subsequent to Binding-- Presumably, MTS-galactoside or MTS-5-galactose reacts directly upon binding. However, the marked increase in the rate of reaction in the presence of Delta <A><AC>&mgr;</AC><AC>&cjs1171;</AC></A>H+ suggests more interesting possibilities. To investigate the problem further, the A122C/C148A mutations were combined with C154G, a mutation that leads to increased binding affinity but almost complete loss of transport activity (42, 43, 44). RSO vesicles with mutant A122C/C148A/C154G exhibit a Kd of about 20 µM, about 10-fold lower than mutant A122C/C148A, as determined by TDG protection against alkylation with NEM (Table I). Relative to mutant A122C/C148A, mutant A122C/C148A/C154G exhibits a significantly greater degree of displacement of bound p-nitrophenyl alpha -D-galactopyranoside (NPG) by TDG as demonstrated by flow dialysis (Fig. 6A), reflecting increased binding affinity. Although not shown, it is highly noteworthy that specificity is maintained, as evidenced by the observation that 10 mM glucose or sucrose has no effect. In contrast, the mutant exhibits little or no active lactose transport (Fig. 6B; Table I). In addition, E. coli HB101 (lacY-lacZ+) expressing the mutant catalyzes almost no downhill translocation of the disaccharide (data not shown).


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Fig. 6.   Effect of C154G (helix V) mutation on MTS-galactoside reactivity of Cys122. A, binding of [3H]NPG to RSO vesicles. Binding of NPG to non-energized vesicles with A122C/C148A or A122C/C148A/C154G at a protein concentration of 32 mg/ml was assayed by flow dialysis. [3H]NPG (840 mCi/mmol) at 15 µM final concentration was added at fraction 1. As indicated by the arrow, TDG at 10 mM final concentration was added at fraction 9 to displace bound NPG. B, initial rates of lactose transport were assayed in vesicles expressing A122C/C148A and A122C/C148A/C154G as indicated. C, rate of reaction of MTS-galactoside or MTS-glucoside with mutant A122C/C148A/C154G determined by blockade of [14C]NEM labeling. RSO membranes were incubated with a given MTS sugar at 200 µM final concentration. The reaction was stopped at the indicated times by dilution, and the samples were washed and labeled with [14C]NEM as described under "Experimental Procedures." The intensity of the radioactive bands corresponding to LacY was quantified with a Storm PhosphorImager and expressed as a percentage obtained in the absence of MTS sugar. D, effect of Delta <A><AC>&mgr;</AC><AC>&cjs1171;</AC></A>H+ on EC50 for MTS-galactoside as detected by blockade of [14C]NEM labeling. NEM labeling after a 10-min treatment with given concentrations of MTS-galactoside in the absence and presence of reduced PMS was quantified with a Storm PhosphorImager and expressed as a percentage obtained in the absence of inactivators. Inset, RSO membranes were incubated with MTS-galactoside at 100 µM final concentration for 10s, and the reaction was stopped by dilution. The membranes were washed and labeled with [14C]NEM for 30 min as described under "Experimental Procedures."

Because mutant A122C/C148A/C154G has essentially no transport activity, reaction of MTS-galactoside with Cys122 was studied by blockade of NEM labeling. Surprisingly, when RSO vesicles are incubated with given concentrations of MTS-galactoside or MTS-glucoside, it is apparent that the mutant essentially loses the ability to distinguish between the MTS sugars; the EC50 is about 60 µM for both (Table I). At 200 µM, there is no detectable difference between the rate of reaction of the mutant with MTS-galactoside and MTS-glucoside (Fig. 6C). Furthermore, similar results are obtained with MTS-5-galactose and MTS-5-glucose (data not shown). Finally, as expected, Delta <A><AC>&mgr;</AC><AC>&cjs1171;</AC></A>H+ has no effect on the reaction of MTS-galactoside (Fig. 6D). Viewed as a whole, the results indicate that MTS-galactoside does not react with the Cys residue at position 122 upon binding but at a subsequent step in the transport cycle.

    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Previous experiments (24) with A122C LacY demonstrate that the galactopyranosyl moiety of disaccharide substrates of LacY is in close proximity to Cys148 in helix V, whereas the non-galactosyl moiety abuts Ala122. On the basis of these observations, we synthesized the MTS sugars described here, demonstrating that both MTS-galactoside and MTS-5-galactose inactivate mutant A122C at least 20 times more rapidly than the corresponding glucose derivatives. Furthermore, it is clear that inactivation of transport is due specifically to reaction of the inactivators with the Cys residue in place of Ala122. Thus, these results presented provide strong confirmation of the previous conclusion (Fig. 7).


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Fig. 7.   LacY binding site with MTS-galactoside as ligand. 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). Cys148 (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). Although LacY with W151F or W151Y transports lactose almost as well as the wild type, the mutants exhibit a 20-50-fold decrease in affinity (45), indicating that the Trp151 stacks with the hydrophobic face of the galactopyranosyl ring, placing it at a right angle with helix IV and abutting Cys148 near position 1. In this orientation, the C-4 OH can H-bond directly with either NH1 or NH2 of Arg144. Because the C-3 OH is close to Glu269 (helix VIII) but at an angle, it is reasonable to suggest that a water molecule may mediate this interaction (50).

One noteworthy aspect of the findings is the difference in potency between the two sets of MTS sugars. Clearly, the ethylene linkage in MTS-galactoside and -glucoside confers greater hydrophobicity than the carbamide linkage in the MTS-5-galactose and -glucose derivatives. The preferential reaction of LacY with hydrophobic thiol reagents (46) is a likely explanation for the differences observed. It may also be relevant that the distance of the reactive thiol in the MTS-5 sugars is significantly further from the C-1 position of the galactopyranosyl ring (Fig. 1).

At first glance, MTS-galactoside and MTS-5-galactose appear to act as straightforward affinity reagents. Both compounds inactivate transport at concentrations at which the corresponding glucose derivatives have no effect. Moreover, when Cys148 is replaced with Ala, Ser, or Asp, there is a qualitative relationship between the respective affinities and the rates of transport and inactivation by MTS-galactoside. In contrast, transport in all three mutants is inactivated at the same rate by MTS-glucoside.

Remarkably, the rate of inactivation by MTS-galactoside and MTS-5-galactose is increased by about 50- and 30-fold, respectively, in the presence of a Delta <A><AC>&mgr;</AC><AC>&cjs1171;</AC></A>H+ (interior negative), and there is no effect of Delta <A><AC>&mgr;</AC><AC>&cjs1171;</AC></A>H+ on inactivation by the corresponding glucose derivatives. There are at least three possible explanations for this finding: (i) Delta <A><AC>&mgr;</AC><AC>&cjs1171;</AC></A>H+ may drive accumulation of the galactose derivatives, thereby leading to an increase in local concentration; (ii) Delta <A><AC>&mgr;</AC><AC>&cjs1171;</AC></A>H+ may increase the accessibility/affinity of the binding site in LacY; or (iii) the galactose derivatives may not react immediately upon binding but at a subsequent step (e.g. a transition intermediate) in the overall transport mechanism induced by binding of the galactosyl analogues and accelerated by Delta <A><AC>&mgr;</AC><AC>&cjs1171;</AC></A>H+. This last interpretation is consistent with the major effect of Delta <A><AC>&mgr;</AC><AC>&cjs1171;</AC></A>H+ on the kinetics of LacY, which is a marked decrease in Km (47).

Although it is possible that MTS-galactoside and MTS-5-galactose are actively transported by LacY, it is unlikely that the marked enhancement of inactivation rates by Delta <A><AC>&mgr;</AC><AC>&cjs1171;</AC></A>H+ is due to accumulation of the inactivators. Thus, at a concentration of 20 µM MTS-galactoside or 100 µM MTS-5-galactose, half-maximal inactivation is observed in less than 5 s (Fig. 4, A and B), whereas significant accumulation of most substrates requires considerably more time.

It is also unlikely that Delta <A><AC>&mgr;</AC><AC>&cjs1171;</AC></A>H+ leads to an increase in accessibility/affinity of the binding site in LacY. Although the data have not been presented here, when the reaction of MTS-galactoside with mutant A122C/C148A is studied by blockade of NEM labeling in inside-out membrane vesicles as a function of MTS-galactoside concentration, there is little or no difference in the concentration of MTS-galactoside that reduces labeling by 50%, and Delta <A><AC>&mgr;</AC><AC>&cjs1171;</AC></A>H+ (interior positive and acid) has no demonstrable effect.

Mutation C154G results in a phenotype that binds ligand but does little or no translocation of lactose across the membrane (42, 43, 44), and the protein appears to be locked in an outwardly facing conformation (44). When this mutation is combined with the A122C/C148A mutations, LacY binds ligand with about 10 times greater affinity than mutant A122C/C148A but catalyzes essentially no lactose translocation. Furthermore, binding specificity is retained, because glucose or sucrose at high concentrations does not displace bound NPG. Surprisingly, however, as judged by NEM labeling (Fig. 4, C and D), mutant A122C/C148A/C154G no longer discriminates between MTS-galactoside and MTS-glucoside, and as expected, there is no effect of Delta <A><AC>&mgr;</AC><AC>&cjs1171;</AC></A>H+. Therefore, it seems apparent that MTS-galactoside, as well as MTS-5-galactose do not react upon binding per se but at a step subsequent to binding. Possibly, a small decrease in distance between position 122 and the MTS moiety at the anomeric position of the galactopyranosyl ring induced by ligand and/or Delta <A><AC>&mgr;</AC><AC>&cjs1171;</AC></A>H+ occurs in order for reaction with the Cys residue at this position to occur (Fig. 7). In any case, it is apparent that LacY turnover is a prerequisite for affinity labeling of the Cys residue at position 122.

    ACKNOWLEDGEMENTS

We thank Yonglin Hu and Vladimir Kasho for the computer graphics shown in Fig. 7 and Gérard Leblanc for kindly providing [3H]NPG. We also thank Arthur Karlin for insisting on the calculation of rate constants.

    FOOTNOTES

* This work was supported in part by National Institutes of Health Grant DK51131-07 and Grant OTKA T34307 from the Hungarian National Research Foundation.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

To whom correspondence should be addressed: 5-748 Macdonald Research Laboratories, Box 951662, Los Angeles, CA 90095-1662. Tel.: 310-206-5053; Fax: 310-206-8623; E-mail: RonaldK@HHMI.UCLA.edu.

Published, JBC Papers in Press, December 5, 2002, DOI 10.1074/jbc.M211355200

2 The concentration of N-ethylmaleimide-reactive Cys residues in right-side-out vesicles is approximately 20 nmol/mg protein (48). Therefore, the concentration of membrane-associated thiol groups per individual reaction mixture is about 60 µM.

    ABBREVIATIONS

The abbreviations used are: LacY, lactose permease; MTS, methanethiosulfonate; TDG, beta -D-galactopyranosyl 1-thio-beta -D-galactopyranoside; NPG, p-nitrophenyl alpha -D-galactopyranoside; NEM, N-ethylmaleimide; Delta <A><AC>&mgr;</AC><AC>&cjs1171;</AC></A>H+, transmembrane proton electrochemical gradient; PMS, phenazine methosulfate; RSO, right-side-out.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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