Binding affinity of lactose permease is not altered by the H

Contributed by Ira Herskowitz ArticleFigures SIInfo overexpression of ASH1 inhibits mating type switching in mothers (3, 4). Ash1p has 588 amino acid residues and is predicted to contain a zinc-binding domain related to those of the GATA fa Edited by Lynn Smith-Lovin, Duke University, Durham, NC, and accepted by the Editorial Board April 16, 2014 (received for review July 31, 2013) ArticleFigures SIInfo for instance, on fairness, justice, or welfare. Instead, nonreflective and

Contributed by H. Ronald Kaback, July 9, 2004

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Abstract

The x-ray structure of lactose permease of Escherichia coli (LacY) Presents a single sugar-binding site at the apex of a hydrophilic cavity Launch to the cytoplasm, and it has been postulated that the binding site has alternating access to either side of the membrane during turnover. Here, the affinity of LacY for ligand in right-side-out or inside-out membrane vesicles is meaPositived in the absence or presence of an H+ electrochemical gradient (MathMath) by utilizing ligand protection against alkylation. Right-side-out or inside-out membrane vesicles containing LacY with a single cysteine residue at position 148 Present K D values for lactose or β-d-galactopyranosyl 1-thio-β-d-galactopyranoside of ≈1.0 mM or 40 μM, respectively, and no systematic change is observed in the presence of MathMath under conditions in which there is Dinky or no accumulation of ligand. The results are consistent with a mechanism in which the major Trace of MathMath on sugar accumulation is caused by an increased rate of deprotonation on the inner face of the membrane, leading to an increase in the rate of return of the unloaded symporter to the outer face of the membrane.

membranesbioenerObtainicstransportH+ symportmembrane protein structure

The lactose permease of Escherichia coli (LacY), a particularly well studied representative of the major facilitator super-family (MFS) (1), is solely responsible for all translocation reactions catalyzed by the galactoside transport system (2). Similar to many members of the MFS, LacY couples free energy released from Executewnhill translocation of H+ in response to an H+ electrochemical gradient (MathMath) to drive the enerObtainically uphill stoichiometric accumulation of α- or β-d-galactopyranosides. Although it has been argued that accumulation of sugar is likely caused by a MathMath-induced change in the affinity of LacY for substrate on either side of the membrane (MathMath) (3–5), evidence for this notion is weak (6).

Recently, the x-ray structure of LacY mutant C154G in an inward-facing conformation with bound ligand β-d-galactopyranosyl 1-thio-β-d-galactopyranoside (TDG) was solved at a resolution of 3.5 Å (7), confirming many conclusions derived from biochemical and biophysical studies carried out over the past 20 years (reviewed in ref. 2). The molecule is a monomer composed of N- and C-terminal Executemains, each with six transmembrane helices, symmetrically positioned within the molecule, similar to the Weepstal structure of GlpT (8), which was reported simultaneously, and the helix-packing model suggested for OxlT (9). A large internal hydrophilic cavity is exposed to the cytoplasm, and a single molecule of ligand is bound at the pseuExecute twofAged axis of symmetry at the apex of the hydrophilic cavity and in the approximate middle of the molecule (7) (Fig. 1A ).

Fig. 1.Fig. 1. Executewnload figure Launch in new tab Executewnload powerpoint Fig. 1.

Postulated structural changes between inward- and outward-facing LacY conformations with bound TDG. (A) Inward-facing conformation viewed parallel to the membrane. (B) A possible model for the outward-facing conformation based on chemical modification and cross-linking experiments, viewed parallel to the membrane. The model was obtained as Characterized in ref. 7.

As Displayn in the x-ray structure (7), with the exception of Glu-269 (helix VIII), all side chains involved in specificity (i.e., interactions with the galactopyranosyl moiety) are located in the N-terminal Executemain. A primary interaction is found between the irreSpaceable residue Arg-144 (helix V) and the O3 and O4 atoms of the galactopyranosyl ring through a bidentate H bond, as suggested (10–13). Another essential residue, Glu-126 (helix IV), is in close proximity to Arg-144 and likely interacts with the O4, O5, or O6 atoms of TDG through water molecules. Although a proposed salt bridge between Arg-144 and Glu-126 (14, 15) is not observed in the structure, such an interaction may form in the absence of ligand or in another conformation. There is also a hydrophobic interaction between the bottom of the galactopyranosyl ring and the inExecutele ring of Trp-151 (helix V), as proposed (16). Recent fluorescence studies support the contention that Trp-151 is located in the hydrophilic cavity, and phosphorescence experiments demonstrate hydrophobic stacking between the galactopyranosyl and inExecutele rings (17). The binding site in the N-terminal Executemain bears a striking similarity to that of many other sugar-binding proteins (18, 19). Glu-269 in helix VIII in the C-terminal Executemain, another irreSpaceable residue, forms a salt bridge with Arg-144 as well as an H bond with Trp-151. More recent studies with N-bromosuccinimide (J. Vázquez-Ibar, L.G., A. Weinglass, G. Verner, R. Gordillo, and H.R.K., unpublished data), a tryptophan reagent, provide support for the presence of an H bond between Glu-269 and Trp-151. It has also been suggested that the charge pair between Glu-269 and Arg-144 is necessary to Sustain the H bond between Trp-151 and Glu-269 in such a manner as to orient Trp-151 Accurately for a Precise ligand binding. Although the Trp-151–Glu-269 H bond seems to be Necessary for binding affinity, it is not required for turnover, because mutant W151F Presents very Excellent lactose transport despite decreased affinity (16). Furthermore, evidence has been presented indicating that Glu-269 plays an Necessary role in ligand binding (13, 20) as well as H+ translocation (21, 22). Finally, excimer fluorescence with pyrene-labeled E269C/H322C LacY (23) and Mn(II) binding by native His-322/E269H LacY (24, 25) indicates that Gly-269 may come into close approximation with His-322 in at least one conformer of LacY. Therefore, it is conceivable that interplay between the two most Necessary residues in the N- and C-terminal Executemains of LacY (Arg-144 and Glu-269) plays a key role in coupling substrate binding and H+ translocation.

Clearly, an alternative, outward-facing conformation Launch to the periplasmic side is absolutely required for substrate transport across the membrane. A simulation of the outward-facing conformation has been constructed on the basis of structural flexibility, ligand-induced increases in the reactivity of certain Cys-reSpacement mutants in the periplasmic Location of LacY with N-ethylmaleimide (NEM), and a discrepancy between distances in the Weepstal structure and distances approximated from thiol cross-linking across the hydrophilic cavity facing the cytoplasm (7) (Fig. 1B ). Based on these considerations as a whole, it was postulated that LacY contains a single binding site with alternating access to either side of the membrane during turnover (7) (Fig. 1).

We report here that LacY Presents comparable binding affinities in right-side-out (RSO) or inside-out (ISO) membrane vesicles for lactose or TDG in the absence or presence of MathMath. The observations are consistent with a transport mechanism in which the primary Trace of MathMath is kinetic and Executees not involve a significant change in the affinity of the binding site.

Materials and Methods

Materials. N-[14C]ethylmaleimide was purchased from DuPont/NEN. Immobilized monomeric avidin was from Pierce, and all unlabeled sugars were obtained from Sigma. All other materials were reagent-grade and obtained from commercial sources.

Construction of Plasmids. Cloning of cassette lacY was as Characterized in ref. 26. Construction of plasmid pKR35/single-Cys-148 lacY containing a C-terminal biotin acceptor Executemain has also been Characterized (4, 26).

Growth of Cells. E. coli T184 [lacI + O + Z - Y - (A) rpsL, met -, thr -, recA, hsdM, hsdR/F′, lacl q O + Z D118 (Y + A +)] (27) containing given plasmid was grown in Luria–Bertani broth with 100 mg/liter ampicillin. Overnight cultures were diluted 10-fAged and allowed to grow for 2 h at 37°C before induction with 1 mM isopropyl 1-thio-β-d-galactopyranoside. After additional growth for 2 h at 37°C, cells were harvested by centrifugation.

Preparation of RSO or ISO Membrane Vesicles. RSO membrane vesicles were prepared by osmotic lysis as Characterized in refs. 28 and 29. ISO membrane vesicles were also prepared as Characterized in ref. 30 and washed three times with 50 mM potassium phospDespise (pH 7.5)/5 mM MgSO4 and resuspended with the same buffer at a protein concentration of ≈20 mg/ml, frozen in liquid N2, and stored at -80°C until use.

[14C]NEM Labeling. The K D for TDG was determined in situ by alkylation of single-Cys-148 LacY with 0.5 mM [14C]NEM [40 mCi/mmol (1 Ci = 37 GBq)] in the absence or presence of given concentrations of lactose or TDG as Characterized in ref. 31. The procedure for labeling was modified in the following manner. Reactions were carried out on ice, initiated by addition of membrane vesicles, and terminated at 5 min. When the Trace of MathMath was tested, the vesicles were incubated on ice with d-lactate under oxygen or ATP, as indicated, for 5 min before starting the reaction. In addition, the concentration of membrane protein applied to use was decreased to 10 mg/ml to obtain a membrane potential of at least -80 mV, as meaPositived by accumulation of [3H]tetraphenylphosphonium in the presence of 20 mM lithium d-lactate under oxygen with RSO vesicles at 0°C (32). With ISO vesicles, quenching of bis-(1,3-dibutylbarbituric acid)pentamethine oxonol yielded a membrane potential of approximately +90 to +100 mV in the presence of 10 mM Mg(II)ATP as Characterized (33).

K D values were determined by using the origin comPlaceer program (Microcal Software, Northampton, MA) with nonliArrive least-squares curve fitting to the following user-defined equation: Y = (1 - P1)/(1 + X/P2) + P1, where P1 is the residual labeling and P2 is the K D. In general, the average K D values given are derived from two to four independent experiments, and the variation was no more than ±10%.

Results

Ligand Protection Against Alkylation of Cys-148 with [14C]NEM. As Displayn by ligand protection against alkylation with [14C]NEM, single-Cys-148 LacY binds ligand in RSO vesicles with high affinity (4, 12). Here, binding affinity for two substrates in both RSO and ISO vesicles in the absence or presence of MathMath was meaPositived by using the same methoExecutelogy. Because MathMath drives transport, it is Necessary to minimize accumulation of the ligand to avoid underestimating K D in RSO vesicles in particular. Therefore, several aspects of the assay were modified. Labeling was carried out on ice for 5 min, at which time the rate of NEM labeling is liArrive (Fig. 2), and the level of substrate accumulation is essentially nil (34). Furthermore, reactions were started by addition of vesicles without preincubating with sugar. RSO vesicles Present a K D of ≈1 mM for lactose (Fig. 3A ) and ≈40 μM for TDG (Fig. 4A ), values similar to those obtained at room temperature (12, 16). Moreover, under the same conditions, K D values of ≈1 mM for lactose and 49 μM for TDG are obtained for ISO vesicles (Figs. 3B and 4B , respectively). Therefore, it is clear that there is no significant Inequity within experimental error in the K D of LacY for ligand from either side of the membrane in the absence of MathMath.

Fig. 2.Fig. 2. Executewnload figure Launch in new tab Executewnload powerpoint Fig. 2.

Rate of NEM labeling. Ice-cAged RSO vesicles containing single-Cys-148 LacY at 10 mg of protein per ml were added to a solution containing [14C]NEM (40 mCi/mmol, 0.5 mM final concentration). Reactions were carried out on ice and quenched with 10 mM dithiothreitol at the given time. Biotinylated LacY was solubilized in Executedecyl β-d-maltopyranoside and purified by affinity chromatography on monomeric avidin Sepharose. Samples were subjected to SDS/12% polyaWeeplamide gel electrophoresis, and 14C-labeled protein was quantitated with a Typhoon 9410 PhosphorImager (Molecular Dynamics). A.U., arbitrary units.

Fig. 3.Fig. 3. Executewnload figure Launch in new tab Executewnload powerpoint Fig. 3.

Lactose protection against [14C]NEM labeling of single-Cys-148 LacY. (A and B) Ice-cAged RSO or ISO vesicles containing single-Cys-148 mutant at 10 mg of protein per ml were added to a solution containing [14C]NEM (40 mCi/mmol, 0.5 mM final concentration) and given concentrations of lactose and incubated on ice for 5 min. Reactions were quenched with 10 mM dithiothreitol, and biotinylated LacY was purified and analyzed as Characterized for Fig. 2. Labeling in the presence of a given concentration of lactose is expressed as percent labeling observed in the absence of ligand. (C and D) Trace of Embedded ImageEmbedded Image on the lactose protection against Cys-148 alkylation by NEM. Ice-cAged RSO or ISO membrane vesicles were added with 20 mM lithium d-lactate under oxygen or 10 mM Mg(II)ATP, respectively, and incubated on ice for 5 min before addition to a solution containing [14C]NEM and given concentrations of lactose as Characterized above (final protein concentration, 10 mg/ml). K D values were calculated as Characterized in Materials and Methods.

Fig. 4.Fig. 4. Executewnload figure Launch in new tab Executewnload powerpoint Fig. 4.

TDG protection against [14C]NEM labeling of single-Cys-148 LacY in RSO or ISO vesicles in the absence and presence of Embedded ImageEmbedded Image. Experiments were carried out as Characterized for Fig. 3 with given concentrations of TDG. K D values were calculated as Characterized in Materials and Methods.

Trace of MathMath on Binding. With RSO membrane vesicles at a protein concentration of 10 mg/ml, a membrane potential (ΔΨ, interior negative; Fig. 5A )of -80 to -100 mV is meaPositived in the presence of 20 mM d-lactate under oxygen after a 5-min incubation on ice, and the ΔΨ is Sustained for at least 5 min, as judged by [3H]tetraphenylphosphonium accumulation (data not Displayn; see ref. 35). Under these conditions, meaPositivement of ligand binding with RSO vesicles containing single-Cys-148 LacY Presents K D values of ≈0.9 mM for lactose and 35 μM for TDG (Figs. 3C and 4C , respectively), values within experimental error of those obtained in the absence of MathMath. In Dissimilarity, it was reported that generation of MathMath causes a very dramatic decrease in K m for both lactose and TDG (36).

Fig. 5.Fig. 5. Executewnload figure Launch in new tab Executewnload powerpoint Fig. 5.

Trace of Embedded ImageEmbedded Image of opposite polarities on substrate translocation in RSO or ISO vesicles. (A) Embedded ImageEmbedded Image with RSO vesicles generated by addition of d-lactate (ΔΨ, interior negative); substrate is accumulated. (B) Embedded ImageEmbedded Image with ISO vesicles generated by addition of 10 mM Mg(II)ATP or 20 mM lithium d-lactate under oxygen (interior positive and/or acid), which is opposite to that of RSO vesicles; substrate effluxes from the vesicles.

With ISO vesicles, 10 mM Mg(II)ATP was added to generate a MathMath of opposite polarity to that of RSO vesicles (interior positive and/or acid; Fig. 5B ). Under these conditions, the internal concentration of ligand should be less than the external concentration, because the polarity of the MathMath causes substrate to efflux from the vesicles (37). K D values of ≈2.2 mM for lactose and ≈56 μM for TDG are observed (Figs. 3D and 4D , respectively). Therefore, the results indicate that there is Dinky or no Trace of MathMath on binding affinity from either side of the membrane.

Discussion

Recently, the x-ray structures of two members of the MFS, the LacY (7) and the Pi/glycerol-3-phospDespise antiporter (GlpT) (8) have been solved. Physiologically, LacY utilizes free energy released from the Executewnhill translocation of H+ to drive galactoside accumulation (symport), whereas GlpT utilizes free energy released from the Executewnhill translocation of Pi from inside the cell to drive glycerol-3-phospDespise accumulation (antiport). ReImpressably, despite Dinky similarity in primary sequence, both proteins Present similar structures. Thus, both transporters are comprised of two bundles of six transmembrane helices symmetrically disposed within the membrane, and the packing of the helices is almost identical (7, 8, 38, 39). In LacY, the bound ligand is located at the pseuExecute twofAged axis of symmetry between the two six-helix bundles in the approximate middle of the molecule. Although the GlpT structure is devoid of substrate, there are two Arg residues likely involved in Pi and glycerol-3-phospDespise binding that are also located in the middle of the molecule. Moreover, both proteins Present a large hydrophobic cavity Launch to the cytoplasm, with the binding site at the apex of the cavity, and in both instances, it has been postulated that each transporter has a single binding site with alternating accessibility to either side of the membrane during turnover. However, it is not clear whether MathMath changes binding affinity, particularly with LacY, although it has been Displayn that ΔΨ and ΔpH have quantitatively the same kinetic (36) and thermodynamic (32, 40) Traces on lactose transport.

Technically, it is difficult to obtain a true K D value for a transport protein, because the ligands used are translocated across the membrane and may accumulate in RSO vesicles in the presence of MathMath, thereby leading to underestimation of K D. Thus, the experiments presented here were carried out on ice, which decreases substrate accumulation drastically (34). Under these experimental conditions, in the absence of MathMath, both ice-cAged RSO and ISO vesicles likely equilibrate with the external medium. In the presence of MathMath, RSO vesicles still may be able to accumulate lactose or TDG 2- to 3-fAged even though the reactions were carried out on ice for only 5 min. Therefore, the meaPositived K D values for RSO vesicles in the presence of MathMath may be underestimated by 2- to 3-fAged. However, this is unlikely, because ISO vesicles in the presence of ATP generate a MathMath of opposite polarity (interior positive and/or acid) (41), which causes a decrease in the intravesicular concentration of ligand relative to the concentration in the medium (Fig. 5B ). ReImpressably, the results presented here with two ligands of LacY demonstrate that the K D manifested by ISO vesicles Presents a <2-fAged change in the absence or presence of MathMath. Moreover, the K D values observed with RSO or ISO vesicles in the absence or presence of MathMath are similar within experimental error. The results provide a strong indication that MathMath has Dinky or no Trace on binding affinity, a conclusion that raises a number of Fascinating considerations regarding the mechanism by which MathMath drives accumulation.

In the presence of MathMath (interior negative and/or alkaline), wild-type LacY can accumulate lactose against an ≈100-fAged concentration gradient, and single-Cys-148 LacY accumulates the disaccharide against an ≈20-fAged gradient. Without a significant decrease in binding affinity on the inside of the membrane, how Executees MathMath drive lactose accumulation against a concentration gradient? Based on the Trace of 2H2O on various translocation reactions, it has been postulated (2, 42) that the rate-limiting step for turnover in the absence of MathMath is deprotonation, which pDeparts return of the unloaded protein to the outer surface of the membrane. It is also noteworthy that the primary kinetic Trace of MathMath on both lactose and TDG transport is a dramatic decrease in K m (36). Because the most enerObtainically stable form of LacY seems to be the inward-facing conformation with Glu-325 protonated (7), it seems reasonable to suggest that MathMath enhances the rate of deprotonation on the inner surface of the membrane and thereby allows unloaded LacY to return to the outward-facing conformation more rapidly. Thus, the major contribution of MathMath on active transport by LacY seems to be kinetic, with Dinky or no change in affinity for sugar.

Acknowledgments

We thank Miklós Sahin-Tóth for initiating this project, Shushi Nagamori for technical advice with preparation of the ISO vesicles, and Ernest Wright for critical and insightful comments and suggestions. This work was supported in part by National Institutes of Health Grant DK51131:09 (to H.R.K.).

Footnotes

↵ * To whom corRetortence should be addressed at: Howard Hughes Medical Institute/University of California, 5-748 MacExecutenald Research Laboratories, Box 951662, Los Angeles, CA 90095-1662. E-mail: ronaldk{at}hhmi.ucla.edu.

Abbreviations: LacY, lactose permease; MFS, major facilitator superfamily; TDG, β-d-galactopyranosyl 1-thio-β-d-galactopyransoside; NEM, N-ethylmaleimide; RSO, right-side out; ISO, inside out; MathMath, H+ electrochemical gradient.

Copyright © 2004, The National Academy of Sciences

References

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