The proton electrochemical gradient induces a kinetic asymmetry in the symport cycle of LacY Xiaoxu Jianga,1, Natalia Ermolovaa, John Lima,2, Seo Woo Choia,3, and H. Ronald Kabacka,b,c,4 aDepartment of Physiology, University of California, Los Angeles, CA 90095; bDepartment of Microbiology, Immunology & Molecular Genetics, University of California, Los Angeles, CA 90095; and cMolecular Biology Institute, University of California, Los Angeles, CA 90095 Contributed by H. Ronald Kaback, November 14, 2019 (sent for review September 24, 2019; reviewed by Phillip E. Klebba and Christopher Miller) LacY catalyzes accumulation of galactosides against a concentration apo conformer was also obtained with LacYww in complex with a + gradient by coupling galactoside and H transport (i.e., symport). nanobody (Nb) bound to the periplasmic side (8). While alternating access of sugar- and H+-binding sites to either side More than a half dozen independent biochemical/spectroscopic of the membrane is driven by binding and dissociation of sugar, the methods combined with the X-ray crystal structures of LacY pro- + ∼ electrochemical H gradient (ΔμH+) functions kinetically by decreas- vide virtually unequivocal evidence that conformational transitions ing the Km for influx 50- to 100-fold with no change in Kd. The affinity between inward- and outward-facing forms result in sugar and + of protonated LacY for sugar has an apparent pK (pKapp)of∼10.5, H transport across the membrane (14). In this manner, cytoplas- due specifically to the pKa of Glu325, a residue that plays an irreplace- mic and periplasmic cavities open/close reciprocally, thereby + + able role in coupling. In this study, rates of lactose/H efflux were allowing alternating exposure of galactoside- and H -binding sites measured from pH 5.0 to 9.0 in the absence or presence of a mem- to either side of the membrane (Fig. 2) (15, 16). Transmembrane brane potential (ΔΨ, interior positive), and the effect of the imposed exchange reactions (i.e., equilibrium exchange and counterflow), ΔΨ on the kinetics of efflux was also studied in right-side-out mem- which reflect alternating access, occur without deprotonation, ∼ ∼ brane vesicles. The findings reveal that ΔμH+ induces an asymmetry and ΔμH+ has no effect on these reactions. Therefore, the driving in the transport cycle based on the following observations: 1) the force for the conformational change(s) responsible for alternating app ∼ ∼ efflux rate of WT LacY exhibits a pK of 7.2 that is unaffected access is not ΔμH+ but binding and dissociation of galactoside (17). by the imposed ΔΨ;2)ΔΨ increases the rate of efflux at all tested The affinity of LacY for galactosides varies with pH, and the pH values, but enhancement is almost 2 orders of magnitude less apparent pK (pKapp) for galactoside binding is unexpectedly al- than observed for influx; 3) mutant Glu325 → Ala does little or no kaline at ∼10.5 (18–21). Direct measurements of Glu325 in situ BIOCHEMISTRY efflux in the absence or presence of ΔΨ, and ambient pH has no by surface enhanced infrared absorption spectroscopy demon- ΔΨ effect; and 4) the effect of (interior positive) on the Km for efflux strate that the side chain has a pKa of 10.5 ± 0.1 (21), which is almost insignificant relative to the 50- to 100-fold decrease in the concurs with the pKapp for galactoside affinity (18, 19). These Km for influx driven by ΔΨ (interior negative). and previous findings (reviewed in refs. 1 and 17) clearly indicate that LacY (i.e., Glu325) is protonated over the physiological pH membranes | transport | permease | membrane proteins | efflux range. Indeed, sugar binding to purified LacY in detergent does scherichia coli lactose permease (LacY), the prototype of the Significance Emajor facilitator superfamily (MFS), catalyzes coupled + + transport of lactose and an H (lactose/H symport). Thus, in Protonation and deprotonation of Glu325 with a pKa of 10.5 is + ∼ + the presence of an electrochemical H gradient (ΔμH+, interior required for symport. Moreover, the H electrochemical gra- ∼ negative and/or alkaline), LacY utilizes free energy generated by dient (ΔμH+) accelerates deprotonation on the intracellular + the energetically downhill flux of H to drive uphill accumulation side with a 50- to 100-fold decrease in the Km. To probe the pK of lactose against a concentration gradient (aka active transport; on the cytoplasmic side of the membrane, rates of lactose/ ∼ + Fig. 1A). Furthermore, in the absence of ΔμH+, downhill transport H efflux were determined from pH 5.0 to 9.0 without or with of lactose in response to a concentration gradient drives uphill flux a membrane potential (ΔΨ, interior positive) in right-side-out + ∼ of H with generation of Δμ +, the polarity of which depends on membrane vesicles. WT lactose efflux has an apparent pK of H ∼ ΔΨ the direction of the lactose gradient (Fig. 1B,influx→ interior 7.2 that is unaffected by , mutant E325A is defective, and ΔΨ ΔΨ positive and acid; Fig. 1C,efflux→ interior negative and alkaline) pH or (interior positive) has no effect. The effect of (interior positive) on the K for efflux with WT LacY is in- (reviewed in ref. 1). m significant relative to the marked effect on influx. LacY is structurally and functionally a monomer (2) with 12 α transmembrane -helices, many of which are shaped irregularly, Author contributions: X.J., N.E., and H.R.K. designed research; X.J., N.E., J.L., and S.W.C. arranged into N- and C-terminal 6-helix bundles with the N and C performed research; X.J., N.E., J.L., S.W.C., and H.R.K. analyzed data; and X.J., N.E., and termini on the cytoplasmic side of the membrane (3). Two 3-helix H.R.K. wrote the paper. inverted repeats are also observed within each 6-helix bundle (4), Reviewers: P.E.K., Kansas State University; and C.M., Howard Hughes Medical Institute and there is a relatively long cytoplasmic loop between helix VI and and Brandeis University. VII that tethers the 2 6-helix pseudosymmetrical domains. Ten The authors declare no competing interest. structures of LacY have been obtained by X-ray crystallography (3, This open access article is distributed under Creative Commons Attribution-NonCommercial- NoDerivatives License 4.0 (CC BY-NC-ND). 5–10). The first, a conformationally restricted mutant C154G, and 1Present address: Department of Chemistry and Physical Sciences, Nicholls State Univer- the WT are inward (cytoplasmic)-open, apo conformers with a sity, Thibodaux, LA 70310. spacious, central aqueous cavity open on the cytoplasmic side and 2Present address: Department of Nutritional Science and Toxicology, University of Cali- tightly sealed on the periplasmic side (3). This conformer appears to fornia, Berkeley, CA 94720. be the resting state of LacY in the membrane (11, 12). A second 3Present address: College of Dentistry, New York University, New York, NY 10010. conformer obtained with double-Trp mutant G46W/G262W 4To whom correspondence may be addressed. Email: [email protected]. (LacYww) (13) is a partially outward (periplasmic)-open, occluded This article contains supporting information online at https://www.pnas.org/lookup/suppl/ conformer with either of 2 bound lactose homologs in the middle of doi:10.1073/pnas.1916563117/-/DCSupplemental. the molecule and a sealed cytoplasmic side (9, 10). An additional First published December 30, 2019. www.pnas.org/cgi/doi/10.1073/pnas.1916563117 PNAS | January 14, 2020 | vol. 117 | no. 2 | 977–981 ∼ involves Glu325 but is hardly affected by ΔμH+, which imposes a strong asymmetry on the symport cycle. Results WT LacY. RSO membrane vesicles expressing WT LacY prepared in KPi (for efflux without an imposed ΔΨ) or NaPi and valino- mycin (for efflux /with an interior positive ΔΨ) were preequili- brated with [14C]lactose at a given pH and rapidly diluted 200- fold into KPi at the same pH, and samples were rapidly filtered ∼ over the initial 40 s in order to measure initial rates of efflux (Fig. Fig. 1. Transport reactions of LacY. (A) ΔμH+-driven influx (i.e., active + 3andSI Appendix,Fig.S2). In the absence of an imposed ΔΨ, transport): free energy released from the downhill translocation of H in ∼ response to Δμ + (interior negative and/or alkaline) generated by the re- efflux is relatively slow at acidic pH and becomes more rapid with H increasing pH. Thus, the half-time for efflux (t ) decreases from spiratory chain or F1/Fo ATPase drives energetically uphill translocation of 1/2 galactoside. (B) Influx: galactoside influx down a concentration gradient ∼62 s at pH 5.0 to ∼22 s at pH 7.0 to ∼11 s at pH 9.0 (Fig. 3 and SI + ∼ Appendix drives uphill H translocation with generation of ΔμH+ (interior positive and ,Fig.S2andTableS1), as indicated previously (34). acidic). (C) Efflux: galactoside efflux down a concentration gradient with Rapid dilution of RSO membrane vesicles preloaded with ∼ generation of ΔμH+ (interior negative and alkaline). (D) Imposition of ΔΨ + NaPi into equimolar KPi in the presence of valinomycin gener- (interior positive) during galactoside efflux: generated by influx of K down ates a membrane potential (ΔΨ, interior positive) that can be a concentration gradient in the presence of valinomycin. monitored by the fluorescence change in bis(1,3-diethylbarbituric acid) [DiBAC4 (3)] (Fig. 1D). The positive ΔΨ causes an increase not induce a change in ambient pH under conditions where in the fluorescence of DiBAC4 (3), which is maintained for at least + SI Appendix binding or release of 1 H /LacY can be measured (20). 1.0 min ( ,Fig.S1). Under these conditions, the rate of + Coupling of galactoside with H translocation is clearly central efflux also increases with pH, and t1/2 decreases (Table 1).