Investigation of sugar binding kinetics of the E. coli sugar/H+ symporter XylE using solid-supported membrane-based electrophysiology.

Bacterial transporters are difficult to study using conventional electrophysiology because of their low transport rates and the small size of bacterial cells. Here, we applied solid-supported membrane-based electrophysiology to derive kinetic parameters of sugar translocation by the Escherichia coli xylose permease (XylE), including functionally relevant mutants. Many aspects of the fucose permease (FucP) and lactose permease (LacY) have also been investigated, which allow for more comprehensive conclusions regarding the mechanism of sugar translocation by transporters of the major facilitator superfamily. In all three of these symporters, we observed sugar binding and transport in real time to determine KM, Vmax, KD, and kobs values for different sugar substrates. KD and kobs values were attainable because of a conserved sugar-induced electrogenic conformational transition within these transporters. We also analyzed interactions between the residues in the available X-ray sugar/H+ symporter structures obtained with different bound sugars. We found that different sugars induce different conformational states, possibly correlating with different charge displacements in the electrophysiological assay upon sugar binding. Finally, we found that mutations in XylE altered the kinetics of glucose binding and transport, as Q175 and L297 are necessary for uncoupling H+ and d-glucose translocation. Based on the rates for the electrogenic conformational transition upon sugar binding (>300 s-1) and for sugar translocation (2 s-1 - 30 s-1 for different substrates), we propose a multiple-step mechanism and postulate an energy profile for sugar translocation. We also suggest a mechanism by which d-glucose can act as an inhibitor for XylE.

Transmembrane serine protease 2 (TMPRSS2) proteolytically activates the epithelial sodium channel (ENaC) by cleaving the channel's γ-subunit.

The epithelial sodium channel (ENaC) is a heterotrimer consisting of α-, ß-, and γ-subunits. Channel activation requires proteolytic release of inhibitory tracts from the extracellular domains of α-ENaC and γ-ENaC; however, the proteases involved in the removal of the γ-inhibitory tract remain unclear. In several epithelial tissues, ENaC is coexpressed with the transmembrane serine protease 2 (TMPRSS2). Here, we explored the effect of human TMPRSS2 on human αßγ-ENaC heterologously expressed in Xenopus laevis oocytes. We found that coexpression of TMPRSS2 stimulated ENaC-mediated whole-cell currents by approximately threefold, likely because of an increase in average channel open probability. Furthermore, TMPRSS2-dependent ENaC stimulation was not observed using a catalytically inactive TMPRSS2 mutant and was associated with fully cleaved γ-ENaC in the intracellular and cell surface protein fractions. This stimulatory effect of TMPRSS2 on ENaC was partially preserved when inhibiting its proteolytic activity at the cell surface using aprotinin but was abolished when the γ-inhibitory tract remained attached to its binding site following introduction of two cysteine residues (S155C-Q426C) to form a disulfide bridge. In addition, computer simulations and site-directed mutagenesis experiments indicated that TMPRSS2 can cleave γ-ENaC at sites both proximal and distal to the γ-inhibitory tract. This suggests a dual role of TMPRSS2 in the proteolytic release of the γ-inhibitory tract. Finally, we demonstrated that TMPRSS2 knockdown in cultured human airway epithelial cells (H441) reduced baseline proteolytic activation of endogenously expressed ENaC. Thus, we conclude that TMPRSS2 is likely to contribute to proteolytic ENaC activation in epithelial tissues in vivo.

A polycystin-2 protein with modified channel properties leads to an increased diameter of renal tubules and to renal cysts.

Mutations in the PKD2 gene cause autosomal-dominant polycystic kidney disease but the physiological role of polycystin-2, the protein product of PKD2, remains elusive. Polycystin-2 belongs to the transient receptor potential (TRP) family of non-selective cation channels. To test the hypothesis that altered ion channel properties of polycystin-2 compromise its putative role in a control circuit controlling lumen formation of renal tubular structures, we generated a mouse model in which we exchanged the pore loop of polycystin-2 with that of the closely related cation channel polycystin-2L1 (encoded by PKD2L1), thereby creating the protein polycystin-2poreL1. Functional characterization of this mutant channel in Xenopus laevis oocytes demonstrated that its electrophysiological properties differed from those of polycystin-2 and instead resembled the properties of polycystin-2L1, in particular regarding its permeability for Ca2+ ions. Homology modeling of the ion translocation pathway of polycystin-2poreL1 argues for a wider pore in polycystin-2poreL1 than in polycystin-2. In Pkd2poreL1 knock-in mice in which the endogenous polycystin-2 protein was replaced by polycystin-2poreL1 the diameter of collecting ducts was increased and collecting duct cysts developed in a strain-dependent fashion.

Asp22 drives the protonation state of the Staphylococcus epidermidis glucose/H+ symporter.

The Staphylococcus epidermidis glucose/H+ symporter (GlcPSe) is a membrane transporter highly specific for glucose and a homolog of the human glucose transporters (GLUT, SLC2 family). Most GLUTs and their bacterial counterparts differ in the transport mechanism, adopting uniport and sugar/H+ symport, respectively. Unlike other bacterial GLUT homologs (for example, XylE), GlcPSe has a loose H+/sugar coupling. Asp22 is part of the proton-binding site of GlcPSe and crucial for the glucose/H+ co-transport mechanism. To determine how pH variations affect the proton site and the transporter, we performed surface-enhanced IR absorption spectroscopy on the immobilized GlcPSe We found that Asp22 has a pKa of 8.5 ± 0.1, a value consistent with that determined previously for glucose transport, confirming the central role of this residue for the transport mechanism of GlcPSe A neutral replacement of the negatively charged Asp22 led to positive charge displacements over the entire pH range, suggesting that the polarity change of the WT reflects the protonation state of Asp22 We expected that the substitution of the residue Ile105 for a serine, located within hydrogen-bonding distance to Asp22, would change the microenvironment, but the pKa of Asp22 corresponded to that of the WT. A167E mutation, selected in analogy to the XylE, introduced an additional protonatable site and perturbed the protonation state of Asp22, with the latter now exhibiting a pKa of 6.4. These studies confirm that Asp22 is the proton-binding residue in GlcPSe and show that charged residues in its vicinity affect the pKa of glucose/H+ symport.

Dawning of a new era in TRP channel structural biology by cryo-electron microscopy.

Cryo-electron microscopy (cryo-EM) permits the determination of atomic protein structures by averaging large numbers of individual projection images recorded at cryogenic temperatures-a method termed single-particle analysis. The cryo-preservation traps proteins within a thin glass-like ice layer, making literally a freeze image of proteins in solution. Projections of randomly adopted orientations are merged to reconstruct a 3D density map. While atomic resolution for highly symmetric viruses was achieved already in 2009, the development of new sensitive and fast electron detectors has enabled cryo-EM for smaller and asymmetrical proteins including fragile membrane proteins. As one of the most important structural biology methods at present, cryo-EM was awarded in October 2017 with the Nobel Prize in Chemistry. The molecular understanding of Transient-Receptor-Potential (TRP) channels has been boosted tremendously by cryo-EM single-particle analysis. Several near-atomic and atomic structures gave important mechanistic insights, e.g., into ion permeation and selectivity, gating, as well as into the activation of this enigmatic and medically important membrane protein family by various chemical and physical stimuli. Lastly, these structures have set the starting point for the rational design of TRP channel-targeted therapeutics to counteract life-threatening channelopathies. Here, we attempt a brief introduction to the method, review the latest advances in cryo-EM structure determination of TRP channels, and discuss molecular insights into the channel function based on the wealth of TRP channel cryo-EM structures.

A Loose Relationship: Incomplete H+/Sugar Coupling in the MFS Sugar Transporter GlcP.

The glucose transporter from Staphylococcus epidermidis, GlcPSe, is a homolog of the human GLUT sugar transporters of the major facilitator superfamily. Together with the xylose transporter from Escherichia coli, XylEEc, the other prominent prokaryotic GLUT homolog, GlcPSe, is equipped with a conserved proton-binding site arguing for an electrogenic transport mode. However, the electrophysiological analysis of GlcPSe presented here reveals important differences between the two GLUT homologs. GlcPSe, unlike XylEEc, does not perform steady-state electrogenic transport at symmetrical pH conditions. Furthermore, when a pH gradient is applied, partially uncoupled transport modes can be generated. In contrast to other bacterial sugar transporters analyzed so far, in GlcPSe sugar binding, translocation and release are also accomplished by the deprotonated transporter. Based on these experimental results, we conclude that coupling of sugar and H+ transport is incomplete in GlcPSe. To verify the viability of the observed partially coupled GlcPSe transport modes, we propose a universal eight-state kinetic model in which any degree of coupling is realized and H+/sugar symport represents only a specific instance. Furthermore, using sequence comparison with strictly coupled XylEEc and similar sugar transporters, we identify an additional charged residue that may be essential for effective H+/sugar symport.

Functional architecture of MFS D-glucose transporters.

The Major Facilitator Superfamily (MFS) is a diverse group of secondary transporters with over 10,000 members, found in all kingdoms of life, including Homo sapiens. One objective of determining crystallographic models of the bacterial representatives is identification and physical localization of residues important for catalysis in transporters with medical relevance. The recently solved crystallographic models of the D-xylose permease XylE from Escherichia coli and GlcP from Staphylococcus epidermidus, homologs of the human D-glucose transporters, the GLUTs (SLC2), provide information about the structure of these transporters. The goal of this work is to examine general concepts derived from the bacterial XylE, GlcP, and other MFS transporters for their relevance to the GLUTs by comparing conservation of functionally critical residues. An energy landscape for symport and uniport is presented. Furthermore, the substrate selectivity of XylE is compared with GLUT1 and GLUT5, as well as a XylE mutant that transports D-glucose.

Substrate-induced changes in the structural properties of LacY.

The lactose permease (LacY) of Escherichia coli, a paradigm for the major facilitator superfamily, catalyzes the coupled stoichiometric translocation of a galactopyranoside and an H(+) across the cytoplasmic membrane. To catalyze transport, LacY undergoes large conformational changes that allow alternating access of sugar- and H(+)-binding sites to either side of the membrane. Despite strong evidence for an alternating access mechanism, it remains unclear how H(+)- and sugar-binding trigger the cascade of interactions leading to alternating conformational states. Here we used dynamic single-molecule force spectroscopy to investigate how substrate binding induces this phenomenon. Galactoside binding strongly modifies kinetic, energetic, and mechanical properties of the N-terminal 6-helix bundle of LacY, whereas the C-terminal 6-helix bundle remains largely unaffected. Within the N-terminal 6-helix bundle, the properties of helix V, which contains residues critical for sugar binding, change most radically. Particularly, secondary structures forming the N-terminal domain exhibit mechanically brittle properties in the unbound state, but highly flexible conformations in the substrate-bound state with significantly increased lifetimes and energetic stability. Thus, sugar binding tunes the properties of the N-terminal domain to initiate galactoside/H(+) symport. In contrast to wild-type LacY, the properties of the conformationally restricted mutant Cys154→Gly do not change upon sugar binding. It is also observed that the single mutation of Cys154→Gly alters intramolecular interactions so that individual transmembrane helices manifest different properties. The results support a working model of LacY in which substrate binding induces alternating conformational states and provides insight into their specific kinetic, energetic, and mechanical properties.

Evolutionary mix-and-match with MFS transporters II.

One fundamentally important problem for understanding the mechanism of coupling between substrate and H(+) translocation with secondary active transport proteins is the identification and physical localization of residues involved in substrate and H(+) binding. This information is exceptionally difficult to obtain with the Major Facilitator Superfamily (MFS) because of the broad sequence diversity of the members. The MFS is the largest and most diverse group of transporters, many of which are clinically important, and includes members from all kingdoms of life. A wide range of substrates is transported, in many instances against a concentration gradient by transduction of the energy stored in an H(+) electrochemical gradient using symport mechanisms, which are discussed herein. Crystallographic structures of MFS members indicate that a deep central hydrophilic cavity surrounded by 12 mostly irregular transmembrane helices represents a common structural feature. An inverted triple-helix structural symmetry motif within the N- and C-terminal six-helix bundles suggests that the proteins may have arisen by intragenic multiplication. In the work presented here, the triple-helix motifs are aligned in combinatorial fashion so as to detect functionally homologous positions with known atomic structures of MFS members. Substrate and H(+)-binding sites in symporters that transport substrates, ranging from simple ions like phosphate to more complex peptides or disaccharides, are found to be in similar locations. It also appears likely that there is a homologous ordered kinetic mechanism for the H(+)-coupled MFS symporters.

Evolutionary mix-and-match with MFS transporters.

Major facilitator superfamily (MFS) transport proteins are ubiquitous in the membranes of all living cells, and ∼25% of prokaryotic membrane transport proteins belong to this superfamily. The MFS represents the largest and most diverse group of transporters and includes members that are clinically important. A wide range of substrates is transported in many instances actively by transduction of the energy stored in an H(+) electrochemical gradient into a concentration gradient of substrate. MFS transporters are characterized by a deep central hydrophilic cavity surrounded by 12 mostly irregular transmembrane helices. An alternating inverted triple-helix structural symmetry within the N- and C-terminal six-helix bundles suggests that the proteins arose by intragenic multiplication. However, despite similar features, MFS transporters share only weak sequence homology. Here, we show that rearrangement of the structural symmetry motifs in the Escherichia coli fucose permease (FucP) results in remarkable homology to lactose permease (LacY). The finding is supported by comparing the location of 34 point mutations in FucP to the location of mutants in LacY. Furthermore, in contrast to the conventional, linear sequence alignment, homologies between sugar- and H(+)-binding sites in the two proteins are observed. Thus, LacY and FucP likely evolved from primordial helix-triplets that formed functional transporters; however, the functional segments assembled in a different consecutive order. The idea suggests a simple, parsimonious chain of events that may have led to the enormous sequence diversity within the MFS.

Apo-intermediate in the transport cycle of lactose permease (LacY).

The lactose permease (LacY) catalyzes coupled stoichiometric symport of a galactoside and an H(+). Crystal structures reveal 12, mostly irregular, transmembrane α-helices surrounding a cavity with sugar- and H(+)- binding sites at the apex, which is accessible from the cytoplasm and sealed on the periplasmic side (an inward-facing conformer). An outward-facing model has also been proposed based on biochemical and spectroscopic measurements, as well as the X-ray structure of a related symporter. Converging lines of evidence demonstrate that LacY functions by an alternating access mechanism. Here, we generate a model for an apo-intermediate of LacY based on crystallographic coordinates of LacY and the oligopeptide/H(+) symporter. The model exhibits a conformation with an occluded cavity inaccessible from either side of the membrane. Furthermore, kinetic considerations and double electron-electron resonance measurements suggest that another occluded conformer with bound sugar exists during turnover. An energy profile for symport is also presented.

An early event in the transport mechanism of LacY protein: interaction between helices V and I.

Helix V in LacY, which abuts and crosses helix I in the N-terminal helix bundle of LacY, contains Arg(144) and Trp(151), two residues that play direct roles in sugar recognition and binding, as well as Cys(154), which is important for conformational flexibility. In this study, paired Cys replacement mutants in helices V and I were strategically constructed with tandem factor Xa protease cleavage sites in the loop between the two helices to test cross-linking. None of the mutants form disulfides spontaneously; however, three mutants (Pro(28) → Cys/Cys(154), Pro(28) → Cys/Val(158) → Cys, and Phe(29) → Cys/Val(158) → Cys) exhibit cross-linking after treatment with copper/1,10-phenanthroline (Cu/Ph) or 1,1-methanediyl bismethanethiosulfonate ((MTS)(2)-1), 3-4 Å), and cross-linking is quantitative in the presence of ligand. Remarkably, with one mutant, complete cross-linking with (MTS)(2)-1 has no effect on lactose transport, whereas quantitative disulfide cross-linking catalyzed by Cu/Ph markedly inhibits transport activity. The findings are consistant with a number of previous conclusions suggesting that sugar binding to LacY causes a localized scissors-like movement between helices V and I near the point where the two helices cross in the middle of the membrane. This ligand-induced movement may act to initiate the global conformational change resulting from sugar binding.

Molecular insights into intra-complex signal transmission during stressosome activation.

The stressosome is a pseudo-icosahedral megadalton bacterial stress-sensing protein complex consisting of several copies of two STAS-domain proteins, RsbR and RsbS, and the kinase RsbT. Upon perception of environmental stress multiple copies of RsbT are released from the surface of the stressosome. Free RsbT activates downstream proteins to elicit a global cellular response, such as the activation of the general stress response in Gram-positive bacteria. The molecular events triggering RsbT release from the stressosome surface remain poorly understood. Here we present the map of Listeria innocua RsbR1/RsbS complex at resolutions of 3.45 Å for the STAS domain core in icosahedral symmetry and of 3.87 Å for the STAS domain and N-terminal sensors in D2 symmetry, respectively. The structure reveals a conformational change in the STAS domain linked to phosphorylation in RsbR. Docking studies indicate that allosteric RsbT binding to the conformationally flexible N-terminal sensor domain of RsbR affects the affinity of RsbS towards RsbT. Our results bring to focus the molecular events within the stressosome complex and further our understanding of this ubiquitous signaling hub.

The Vibrio vulnificus stressosome is an oxygen-sensor involved in regulating iron metabolism.

Stressosomes are stress-sensing protein complexes widely conserved among bacteria. Although a role in the regulation of the general stress response is well documented in Gram-positive bacteria, the activating signals are still unclear, and little is known about the physiological function of stressosomes in the Gram-negative bacteria. Here we investigated the stressosome of the Gram-negative marine pathogen Vibrio vulnificus. We demonstrate that it senses oxygen and identified its role in modulating iron-metabolism. We determined a cryo-electron microscopy structure of the VvRsbR:VvRsbS stressosome complex, the first solved from a Gram-negative bacterium. The structure points to a variation in the VvRsbR and VvRsbS stoichiometry and a symmetry breach in the oxygen sensing domain of VvRsbR, suggesting how signal-sensing elicits a stress response. The findings provide a link between ligand-dependent signaling and an output - regulation of iron metabolism - for a stressosome complex.

Limited reversibility of transmembrane proton transfer assisting transmembrane electron transfer in a dihaem-containing succinate:quinone oxidoreductase.

Membrane protein complexes can support both the generation and utilisation of a transmembrane electrochemical proton potential (Deltap), either by supporting transmembrane electron transfer coupled to protolytic reactions on opposite sides of the membrane or by supporting transmembrane proton transfer. The first mechanism has been unequivocally demonstrated to be operational for Deltap-dependent catalysis of succinate oxidation by quinone in the case of the dihaem-containing succinate:menaquinone reductase (SQR) from the Gram-positive bacterium Bacillus licheniformis. This is physiologically relevant in that it allows the transmembrane potential Deltap to drive the endergonic oxidation of succinate by menaquinone by the dihaem-containing SQR of Gram-positive bacteria. In the case of a related but different respiratory membrane protein complex, the dihaem-containing quinol:fumarate reductase (QFR) of the epsilon-proteobacterium Wolinella succinogenes, evidence has been obtained that both mechanisms are combined, so as to facilitate transmembrane electron transfer by proton transfer via a both novel and essential compensatory transmembrane proton transfer pathway ("E-pathway"). Although the reduction of fumarate by menaquinol is exergonic, it is obviously not exergonic enough to support the generation of a Deltap. This compensatory "E-pathway" appears to be required by all dihaem-containing QFR enzymes and results in the overall reaction being electroneutral. However, here we show that the reverse reaction, the oxidation of succinate by quinone, as catalysed by W. succinogenes QFR, is not electroneutral. The implications for transmembrane proton transfer via the E-pathway are discussed.

The correlation of cathodic peak potentials of vitamin K(3) derivatives and their calculated electron affinities. The role of hydrogen bonding and conformational changes.

2-methyl-1,4-naphtoquinone 1 (vitamin K(3), menadione) derivatives with different substituents at the 3-position were synthesized to tune their electrochemical properties. The thermodynamic midpoint potential (E(1/2)) of the naphthoquinone derivatives yielding a semi radical naphthoquinone anion were measured by cyclic voltammetry in the aprotic solvent dimethoxyethane (DME). Using quantum chemical methods, a clear correlation was found between the thermodynamic midpoint potentials and the calculated electron affinities (E(A)). Comparison of calculated and experimental values allowed delineation of additional factors such as the conformational dependence of quinone substituents and hydrogen bonding which can influence the electron affinities (E(A)) of the quinone. This information can be used as a model to gain insight into enzyme-cofactor interactions, particularly for enzyme quinone binding modes and the electrochemical adjustment of the quinone motif.

CopR, a Global Regulator of Transcription to Maintain Copper Homeostasis in Pyrococcus furiosus.

Although copper is in many cases an essential micronutrient for cellular life, higher concentrations are toxic. Therefore, all living cells have developed strategies to maintain copper homeostasis. In this manuscript, we have analyzed the transcriptome-wide response of Pyrococcus furiosus to increased copper concentrations and described the essential role of the putative copper-sensing metalloregulator CopR in the detoxification process. To this end, we employed biochemical and biophysical methods to characterize the role of CopR. Additionally, a copR knockout strain revealed an amplified sensitivity in comparison to the parental strain towards increased copper levels, which designates an essential role of CopR for copper homeostasis. To learn more about the CopR-regulated gene network, we performed differential gene expression and ChIP-seq analysis under normal and 20 µM copper-shock conditions. By integrating the transcriptome and genome-wide binding data, we found that CopR binds to the upstream regions of many copper-induced genes. Negative-stain transmission electron microscopy and 2D class averaging revealed an octameric assembly formed from a tetramer of dimers for CopR, similar to published crystal structures from the Lrp family. In conclusion, we propose a model for CopR-regulated transcription and highlight the regulatory network that enables Pyrococcus to respond to increased copper concentrations.

Recent progress on obtaining theoretical and experimental support for the "E-pathway hypothesis" of coupled transmembrane electron and proton transfer in dihaem-containing quinol:fumarate reductase.

Reconciliation of apparently contradictory experimental results obtained on the quinol: fumarate reductase (QFR), a dihaem-containing respiratory membrane protein complex from Wolinella succinogenes, was previously obtained by the proposal of the so-called E-pathway hypothesis. According to this hypothesis, transmembrane electron transfer via the haem groups is strictly coupled to co-transfer of protons via a transiently established, novel pathway, proposed to contain the side chain of residue Glu-C180 and the distal haem ring-C propionate as the most prominent components. This hypothesis has recently been supported by both theoretical and experimental results. Multiconformation continuum electrostatics calculations predict Glu-C180 to undergo a combination of proton uptake and conformational change upon haem reduction. Strong experimental support for the proposed role of Glu-C180 in the context of the "E-pathway hypothesis" is provided by the effects of replacing Glu-C180 with Gln or Ile by site-directed mutagenesis, the consequences of these mutations for the viability of the resulting mutants, together with the structural and functional characterisation of the corresponding variant enzymes, and the comparison of redox-induced Fourier-transform infrared (FTIR) difference spectra for the wild type and Glu-C180-->Gln variant. A possible haem propionate involvement has recently been supported by combining (13)C-haem propionate labelling with redox-induced FTIR difference spectroscopy.

Molecular insights into lipid-assisted Ca2+ regulation of the TRP channel Polycystin-2.

Polycystin-2 (PC2), a calcium-activated cation TRP channel, is involved in diverse Ca2+ signaling pathways. Malfunctioning Ca2+ regulation in PC2 causes autosomal-dominant polycystic kidney disease. Here we report two cryo-EM structures of distinct channel states of full-length human PC2 in complex with lipids and cations. The structures reveal conformational differences in the selectivity filter and in the large exoplasmic domain (TOP domain), which displays differing N-glycosylation. The more open structure has one cation bound below the selectivity filter (single-ion mode, PC2SI), whereas multiple cations are bound along the translocation pathway in the second structure (multi-ion mode, PC2MI). Ca2+ binding at the entrance of the selectivity filter suggests Ca2+ blockage in PC2MI, and we observed density for the Ca2+-sensing C-terminal EF hand in the unblocked PC2SI state. The states show altered interactions of lipids with the pore loop and TOP domain, thus reflecting the functional diversity of PC2 at different locations, owing to different membrane compositions.

pH Regulation of Electrogenic Sugar/H+ Symport in MFS Sugar Permeases.

Bacterial sugar symporters in the Major Facilitator Superfamily (MFS) use the H+ (and in a few cases Na+) electrochemical gradients to achieve active transport of sugar into the cell. Because a number of structures of MFS sugar symporters have been solved recently, molecular insight into the transport mechanism is possible from detailed functional analysis. We present here a comparative electrophysiological study of the lactose permease (LacY), the fucose permease (FucP) and the xylose permease (XylE), which reveals common mechanistic principles and differences. In all three symporters energetically downhill electrogenic sugar/H+ symport is observed. Comparison of the pH dependence of symport at symmetrical pH exhibits broad bell-shaped pH profiles extending over 3 to 6 pH units and a decrease at extremely alkaline pH ≥ 9.4 and at acidic to neutral pH = 4.6-7.5. The pH dependence can be described by an acidic to neutral apparent pK (pKapp) and an alkaline pKapp. Experimental evidence suggests that the alkaline pKapp is due to H+ depletion at the protonation site, while the acidic pKapp is due to inhibition of deprotonation. Since previous studies suggest that a single carboxyl group in LacY (Glu325) may be the only side chain directly involved in H+ translocation and a carboxyl side chain with similar properties has been identified in FucP (Asp46) and XylE (Asp27), the present results imply that the pK of this residue is switched during H+/sugar symport in all three symporters.