Monitoring conformational rearrangements in the substrate-binding site of a membrane transport protein by mass spectrometry.

Combined biochemical, biophysical, and crystallographic studies on the lactose permease of Escherichia coli suggest that Arg-144 (helix V) forms a salt bridge with Glu-126 (helix IV), which is broken during substrate binding, thereby permitting the guanidino group to form a bidentate H-bond with the C-4 and C-3 O atoms of the galactopyranosyl moiety and an H-bond with Glu-269 (helix VIII). To examine the relative interaction of Arg-144 with these two potential salt bridge partners (Glu-126 and Glu-269) in the absence of substrate, the covalent modification of the guanidino group was monitored with the Arg-specific reagent butane-2,3-dione using electrospray ionization mass spectrometry. In a functional background, the reactivity of Arg-144 with butane-2,3-dione is low ( approximately 25%) and is reduced by a factor of approximately 2 by preincubation with ligand. Interestingly, although replacement of Glu-126 with Ala results in a 3-fold increase in the reactivity of Arg-144, replacement of Glu-269 with Ala elicits virtually no effect. Taken together, these results suggest that in the absence of substrate the interaction between Arg-144 and Glu-126 is much stronger than the interaction with Glu-269, supporting the contention that sugar recognition leads to rearrangement of charge-paired residues essential for sugar binding.

The lactose permease of Escherichia coli (LacY) is a paradigm for members of the MFS (2), which utilize free energy from an electrochemical proton gradient to catalyze the energetically uphill transport of D-galactopyranosides across the cell membrane or vice versa (3). Typical of the MFS, LacY contains 12 transmembrane helices connected by hydrophilic loops, with both the N and C termini on the cytoplasmic face of the membrane (4 -7), and it is functionally and structurally a monomer (7)(8)(9).
LacY is selective for disaccharides containing a D-galactopyranosyl ring as well as D-galactose (10 -12) but does not interact with D-glucopyranosides or D-glucose (12)(13)(14). Therefore, the substrate specificity of LacY is directed toward the galactopyranosyl ring of substrate and, most importantly, toward the C-4 OH of the galactopyranosyl ring. Galactose is the most specific substrate of LacY but has a very low affinity (K d(app) ϳ 30 mM) (12), and substitutions at the anomeric position can markedly increase affinity up to 3 orders of magnitude by nonspecific interactions (15).
An important advance with regard to unraveling the mechanism of LacY has been the solution of the x-ray structure of the inward facing conformation of a LacY mutant (C154G) with a bound lactose homologue, ␤-D-galactopyranosyl-1-thio-␤-Dgalactopyranoside (TDG) (7). The overall structure reveals pseudo 2-fold symmetry between the N-and the C-terminal 6-helix domains, as proposed for other members of the MFS (16). Remarkably, similar pseudo symmetry and helical packing were also found simultaneously in the x-ray structure of the P i /glycerol-3-phosphate (GlpT) antiporter of E. coli (17), another MFS member. This structural similarity is even more interesting because the two proteins have only ϳ20% homology and catalyze completely different reactions. Interestingly, LacY and GlpT were crystallized in the same inward facing conformation, although the GlpT structure was obtained in the absence of bound ligand.
The sugar-binding site in LacY is located in a large waterfilled cavity open only to the cytoplasm at the pseudo 2-fold axis of symmetry situated in the approximate middle of the membrane (Fig. 1A). Arg-144 (helix V) and Glu-126 (helix IV) are the major determinants for sugar binding (Fig. 1B). Arg-144 determines the stereoselectivity of LacY forming a bidentate H-bond with the O-3 and O-4 atoms of the galactopyranosyl ring, whereas Glu-126 probably interacts with O-4, O-5, or O-6 via water molecules. Interestingly, however, indirect biochemical approaches suggest the presence of a salt bridge between Glu-126 and Arg-144 when substrate is not bound (18 -22). Furthermore, as predicted (23), the structure exhibits hydrophobic stacking between the galactopyranosyl ring of TDG and the indole side chain of Trp-151 (Fig. 1B). The nature of this interaction has been extended recently (24) by luminescence spectroscopy.
Glu-269 also plays an important role in the architecture of the binding site in the inward facing conformation (Fig. 1B), and functional characterization of Glu-269 mutants indicates that this may be a key residue in coupling between sugar binding and H ϩ translocation (7,19,(25)(26)(27)(28)(29). Thus, with the exception of aspartic acid, which demonstrates a dramatic alteration in the H ϩ /TDG stoichiometry (25), all other replacements for Glu-269 do not bind sugar and are completely defective in all modes of translocation (28). In the current structure, Glu-269 forms a salt bridge with Arg-144 and is situated in a position to form an H-bond with the indole nitrogen of Trp-151. Finally, studies combining chemical modification with mass spectrometry suggest an interaction between Glu-269 and the O-3 of p-nitrophenyl-␣-D-galactopyranoside (NPGal), probably mediated by a water molecule (30). From the combined data, it seems clear that understanding the role of Glu-269 in the structural organization of the binding site is essential to understand coupling between substrate and H ϩ translocation.
In this paper, we present experiments that describe the interplay among Glu-126, Arg-144, and Glu-269 in sugar recognition. Experimentally, this is accomplished by engineering LacY to retain function and simultaneously produce a unique peptide containing Arg-144 on cleavage with cyanogen bromide (CNBr). Subsequently, the effects of substrate and mutations on the reactivity of Arg-144 can be probed using the Argspecific reagent butane-2,3-dione (BD) (31) and monitored by ESI-MS. Consistent with the x-ray structure, substrate protects against BD modification of Arg-144. Moreover, when Glu-126, the putative charge pair partner of Arg-144, is replaced with Ala, the reactivity of Arg-144 with BD increases ϳ3-fold. Remarkably, replacement of another carboxyl group close to Arg-144 and within 5 Å of the substrate (E269A) elicits no significant effect on the reactivity of Arg-144. Taken together, the observations provide further evidence for a salt bridge between Glu-126 and Arg-144 that is broken during sugar binding, a process involving the rearrangement of interactions between essential residues.
Construction of LacY Mutants-Two-step PCR mutagenesis of the genes encoding wild-type and C154G LacY (in plasmid pT7-5 bearing a His 10 tag) generated products containing the double mutation R135M/ R142S. These PCR products were subcloned back into wild-type LacY as PstI/KpnI fragments. Thereafter, PCR fragments containing the mutations E126A, E269A, and E126A/E269A were generated on the R135M/R142S and R135M/R142S/C154G backgrounds and subcloned back into wild-type LacY in pT7-5 as PstI/SpeI fragments. All constructs were confirmed by DNA sequencing.
Transport Assays-E. coli T184 expressing given permease mutants were washed once with 100 mM potassium P i (pH 7.5), 10 mM MgSO 4 and adjusted to an optical density of 10.0 at 600 nm (0.7 mg of protein/ ml). Transport was initiated by the addition of  (10), and E126A/E269A/R135M/R142S/C154G (11). Aliquots (50 l) of cell suspensions containing 35 g of protein in 100 mM potassium P i (pH 7.5), 10 mM MgSO 4 were assayed at 0.4 mM final external concentration of lactose as described under "Experimental Procedures." mmol) to a final concentration of 0.4 mM. Samples were quenched at given times with 100 mM potassium P i (pH 5.5), 100 mM LiCl and assayed by rapid filtration as described previously (32).
Bacterial Strains, Growth, and LacY Purification-E. coli T184 (lacZ Ϫ Y Ϫ ) transformed with pT7-5 encoding LacY mutants with a His 10 tag was grown in 6 liters of Luria-Bertani broth at 37°C, respectively, containing ampicillin (100 g/ml) to an A 600 of 0.6 and induced with 0.5 mM isopropyl-1-thio-␤-D-galactopyranoside. Cells were disrupted by passage through a French pressure cell, and the membrane fraction was harvested by ultracentrifugation. Membranes were solubilized by DDM to a final concentration of 2%, and LacY was purified by cobalt affinity chromatography (Talon Superflow TM , Palo Alto, CA) as described previously (7). Protein eluted with 200 mM imidazole was dialyzed against 50 mM sodium P i (pH 7.5), 100 mM NaCl, and 0.02% DDM, concentrated by using a Vivaspin 20 concentrator (30-kDa cutoff) (Vivascience, Hannover, Germany), and stored on ice. As determined by SDS-12% PAGE followed by silver nitrate staining, the preparations contained only a single band with an molecular mass of ϳ33 kDa. Protein was assayed by using a micro-BCA kit (Pierce).
Fluorescence Spectroscopy-Fluorescence was recorded by a Fluorolog 3 spectrofluorimeter (Spex Industries, Edison, NJ) equipped with a double grating monochromator, Glan Thompson polarizers, and a cuvette holder thermostatted at 25°C. Time courses of 2-(4-maleimidoanalino)naphthalene-6-sulfonic acid labeling were conducted with 1 M protein in 50 mM sodium P i (pH 7.5), 100 mM NaCl, and 0.02% DDM in 1 ϫ 1-cm quartz cuvettes with constant stirring at 25°C. Reactions were initiated by adding 2-(4-maleimidoanalino)naphthalene-6-sulfonic acid to a final concentration of 5 M, and fluorescence was recorded over time. Excitation and emission wavelengths were 332 and 415 nm, respectively, and slits were 2 nm in both monochromators.
DiPC, BD Labeling, and Sample Preparation for Reverse Phase HPLC of CNBr Fragments-DiPC and BD were freshly prepared as 1 M stocks dissolved in Me 2 SO. After dialysis of purified protein in 50 mM sodium P i (pH 6.0), 100 mM NaCl, 0.02% DDM (for DiPC modification) or 50 mM NaBO 2 (pH 8.0), 100 mM NaCl, and 0.02% DDM (for BD modification), the protein was reacted with DiPC or BD at 30°C for a given time and immediately precipitated with CHCl 3 /MeOH, as described previously (33). One aliquot (100 l) of the aqueous protein solution at a concentration of 1-2 mg/ml was diluted 1:3 (v/v) with MeOH and mixed briefly. CHCl 3 (100 l) was added and mixed, yielding a single phase. Phase separation was accomplished by adding water (200 l) and mixing vigorously. The phases were separated by centrifugation (10,000 ϫ g for 2 min), yielding a precipitate at the interface. The bulk of the upper aqueous methanol phase was then carefully aspirated, and methanol (300 l) was added. After gently mixing, insoluble protein in the single phase mixture was recovered by centrifugation at 10,000 ϫ g for 1 min. For cleavage of the protein, after drying, the pellet was resuspended in a saturated solution of CNBr in 90% formic acid (34) and left for 2 h in the dark. Formic acid was removed in a vacuum centrifuge, and the pellet was left overnight in 0.5 ml of 0.1% trifluoroacetic acid in water. The sample was dried again and resuspended in 60% formic acid immediately prior to RP-HPLC.
HPLC-For the final purification of LacY after covalent modification with DiPC or BD, HPLC was used prior to ESI-MS in an in-line setup. To separate the CNBr peptides generated from LacY, a polystyrene/ divinylbenzene co-polymer column (5 m, 300 Å, 150 ϫ 2.1 mm) (PLRP/S, Polymer Labs) at 40°C was used for reverse phase chromatography (35). Following equilibration in a mixture of 95% solvent A (0.1% trifluoroacetic acid in water) to 5% solvent B (0.1% trifluoroacetic acid in CH 3 CN:isopropanol (1:1)) for 5 min, the percentage of solvent B was increased to 40% over the next 25 min and further to 100% over the subsequent 120 min at a flow rate of 0.1 ml/min. All chromatographic separations were performed at 40°C using a modified ABI 120A dual syringe pump HPLC equipped with a post-detector (A 280 ) splitter for backpressure regulation.
Electrospray Ionization Mass Spectrometry-ESI-MS was performed using a PerkinElmer Life Sciences Sciex API III triple quadrupole instrument operating in the positive ion mode as described previously (36). The orifice potential was ramped from 60 to 120 V across the mass range (600 -2300 a.m.u.) for CNBr fragments. Tandem mass spectrometry fragment ion spectra were obtained by splitting the flow into the API III during RP-HPLC and collecting peptides of interest for infusion (3 l/min) into a ThermoFinnigan LCQ Deca ion trap instrument with a 33-gauge stainless steel needle source at 3.3 kV. The nomenclature used for fragment ions is N-terminal fragments (b type) and C-terminal fragments (y type) (37,38).

Engineering LacY to Probe the Reactivity of Arg-144 -
The group-specific modification reagent BD reacts specifically with the guanidino group of Arg enabling the environment of Arg-144 to be probed in a functional background (see Fig. 3A). E. coli T184 (lacZ Ϫ Y Ϫ ) expressing wild-type LacY catalyzes lactose accumulation at a high rate to a steady state of ϳ150 nmol/mg of protein in ϳ10 min, which corresponds to a 100-fold concentration gradient ( Fig. 2A). Similarly, mutant R135M/ R142S demonstrates a similar rate and steady-state level of lactose accumulation, although mutants E126A/R135M/R142S, E269A/R135M/R142S, and E269A/E126A/R135M/R142S do not significantly accumulate lactose to levels higher than T184 transformed with a vector lacking the LacY gene ( Fig. 2A). Like mutant C154G (40), mutant R135M/R142S/C154G demonstrates a very low level of lactose accumulation and, as expected, mutants E126A/R135M/R142S/C154G, E269A/R135M/ R142S/C154G, and E269A/E126A/R135M/R142S/C154G do not accumulate lactose (Fig. 2B) (Fig. 3B, I). BD reacts with mutant R135M/R142S/C154G, and cleavage with CNBr reveals that approximately 20% of the peptide 135 SNFEFGSARM 145 is modified rapidly; however, the reaction is completed at ϳ25% mod- ification in 20 min, most likely because of instability in water (Fig. 3C). The modified species has an m/z value of 592.2 for the doubly charged peptide and a measured monoisotopic mass of 1182.2 compared with the expected m/z and monoisotopic values of 592.25 and 1183.5, respectively. Collision-activated dissociation of the peptide reveals fragment ions consistent with the sequence 135 SNFEFGSARM 145 modified at Arg-144 by BD (Fig. 3B, II).
Following reaction of mutant R135M/R142S/C154G with BD for 30 min, 33% of peptide 135 SNFEFGSARM 145 is modified (Fig. 4, compare I with II). Preincubation with the high affinity ligand NPGal reduces the rate of modification of the decapeptide 135 SNFEFGSARM 145 by over 2-fold (Fig. 4, compare II with III). The effect is substrate-specific because NPGlc, which has no measurable affinity for LacY, has no significant effect on the rate of BD modification (data not shown). Interestingly, with mutant E126A/R135M/R142S/C154G, the decapeptide 135 SNFEFGSARM 145 is modified by BD to over twice the extent of that observed with R135M/R142S/C154G (Fig. 4, compare V with II), and preincubation with NPGal has no effect whatsoever on BD modification (Fig. 4, V and VI).
Because replacing Glu-126 with Ala elicits a marked effect on the extent of BD reactivity with Arg-144, the reactivity of another residue involved in binding (Glu-269) was probed (30). By this means, it can be determined whether the E126A mutation elicits a widespread conformational change in the protein or a localized alteration. As shown previously, reaction of mutant R135M/R142S/C154G with the carboxyl-specific reagent DiPC leads to covalent modification of ϳ33% of the peptide 268 GELLNASIM 276 in 30 min (Fig. 5, compare I with II). Preincubation with NPGal reduces the rate of modification of 268 GELLNASIM 276 by ϳ2-fold (Fig. 5, compare II with III). The effect is substrate-specific because NPGlc, which has no measurable affinity for LacY, exerts no significant effect on the rate of DiPC modification (data not shown). In mutant E126A/ R135M/R142S/C154G, 268 GELLNASIM 276 is modified by DiPC at approximately the same rate (ϳ30% in 30 min) (Fig. 5, compare IV with V); however, in this mutant, preincubation with NPGal has little effect on DiPC modification (Fig. 5, compare V and VI).
Influence of Position 269 on the Reactivity of Arg-144 -The x-ray structure of LacY indicates that Arg-144 forms a salt bridge with Glu-269 in the presence of substrate. Therefore, the influence of Glu-269 on the reactivity of Arg-144 with BD was examined in the absence of substrate in mutants E269A/ R135M/R142S/C154G and E269A/E126A/R135M/R142S/C154G.
Following reaction of mutant E269A/R135M/R142S/C154G with BD for 30 min, ϳ30% of peptide 135 SNFEFGSARM 145 is modified (Fig. 6, compare I with II). Preincubation with the high affinity ligand NPGal has no significant effect on BD modification of the decapeptide 135 SNFEFGSARM 145 (Fig. 6, compare II with III). Likewise, with mutant E269A/E126A/ R135M/R142S/C154G, the decapeptide 135 SNFEFGSARM 145 is modified by BD to over twice the extent (ϳ66%) of that observed with E269A/R135M/R142S/C154G (Fig. 6, compare V with II), and preincubation with NPGal has no significant effect on BD modification (Fig. 6, V and VI). These differences in reactivity between mutants are constant over a time course (Fig. 7). DISCUSSION The x-ray structure of an inward facing conformation of LacY with bound substrate (7) provides a framework for understanding the molecular basis of substrate recognition and translocation by LacY, a paradigm for the MFS family of secondary membrane transport proteins. By integrating the structure with biochemical and biophysical analyses, a model for the mechanism for lactose/H ϩ symport has been proposed (7). Influx consists of six steps starting from the outward facing conformation as shown in sights into local alterations in structure induced by substrate binding.
One surprise from the x-ray structure is the absence of a salt bridge between Glu-126 and Arg-144. Previous functional studies demonstrate that the carboxyl and guanidino side chains at positions 126 and 144, respectively, are critical for substrate binding (19,20) and translocation (41). Furthermore, close physical proximity of Glu-126 and Arg-144 has been inferred from site-directed spectroscopic studies with E126C/R144C LacY (42) and spontaneous disulfide formation (22). Finally, the reactivity of Cys-148 (helix V) is reduced dramatically by removal of the carboxyl group from position 126 (19). However, interpretation of all of these experiments must be placed within the context of either an inactive background (E126C/R144C) or changes in reactivity of an indirect reporter (Cys-148).
In this study, by exploiting chemical modification and the ability of HPLC in conjunction with mass spectrometry to resolve complex mixtures, the environment of Arg-144 is analyzed directly on a functional background that permits analysis of substrate effects on native irreplaceable side chains. In a functional background, in which Arg-144 and Glu-126 are likely charge-paired, the Arg-specific reagent BD reacts with Arg-144. Furthermore, preincubation with a substrate analog reduces the rate of modification by a factor of two. However, when the charge pair is disrupted (E126A), the reactivity of Arg-144 with BD increases markedly. Two findings indicate that the interaction between Glu-126 and Arg-144 is being monitored primarily; (i) E126A causes a relatively large change in Arg-144 reactivity, but mutant E269A has little or no effect, and (ii) E126A causes essentially no change in the reactivity of Glu-269.
From a mechanistic perspective, one interesting feature of the x-ray structure (Fig. 1B) is the relative proximity of Arg-144 to both Glu-126 (ϳ5.1 Å) and Glu-269 (ϳ4.7 Å) in the substrate-bound form. Thus, in the absence of substrate, it is notable that the E269A mutation leads to no significant increase in the reactivity of Arg-144 with BD, showing that the interaction of Arg-144 is significantly stronger with Glu-126 rather than with Glu-269. Hence, in the absence of substrate, Arg-144 forms a salt bridge with Glu-126 and does not significantly interact with Glu-269; however, on substrate binding, the salt bridge between Glu-126 and Arg-144 breaks in a coordinated manner as part of an integrated series of events leading to binding and translocation. Although these observations are made in the mutant C154G background that favors the inward facing conformation (Fig. 8, compare step IV, with steps V and VI), it is likely that substrate recognition in the outward facing conformation involves the same conformational changes (Fig. 8, compare steps II and III). In other words, sugar binding to LacY may involve a dynamic rearrangement of the interactions involved in substrate binding coupled with a conformational transition to the inward facing conformation.