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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.

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.

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.

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.

Reconciliation of apparently contradictory experimental results obtained on the quinol:fumarate reductase, a diheme-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 heine groups is strictly coupled to cotransfer of protons via a transiently established pathway thought to contain the side chain of residue Glu-C180 as the most prominent component. Here we demonstrate that, after replacement of Glu-C180 with Gin or lie by site-directed mutagenesis, the resulting mutants are unable to grow on fumarate, and the membrane-bound variant enzymes lack quinol oxidation activity. Upon solubilization, however, the purified enzymes display 1/10 of the specific quinol oxidation activity of the wild-type enzyme and unchanged quinol Michaelis constants, [K.sub.m]. The refined x-ray crystal structures at 2.19 [Angstrom] and 2.76 [Angstrom] resolution, respectively, rule out major structural changes to account for these experimental observations. Changes in the oxidation-reduction heme midpoint potential allow the conclusion that deprotonation of Glu-C180 in the wild-type enzyme facilitates the reoxidation of the reduced high-potential heine. Comparison of solvent isotope effects indicates that a rate-limiting proton transfer step in the wild-type enzyme is lost in the Glu-C180 [right arrow] Gin variant. The results provide experimental evidence for the validity of the E pathway hypothesis and for a crucial functional role of Glu-C180. anaerobic respiration | atomic model | bioenergetics | membrane protein | succinate:quinone oxidoreductase

The purification, reconstitution into proteoliposomes and functional characterization of the diheme-containing succinate:menaquinone reductase from the Gram-positive bacterium Bacillus licheniformis is described. The design, synthesis and characterization of quinones with finely tuned oxidation/reduction potentials have provided evidence for [DELTA]p (transmembrane electrochemical proton potential)-dependent catalysis of succinate oxidation by quinone as well as for [DELTA]p generation upon catalysis of fumarate reduction by quinol.

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. [ABSTRACT FROM AUTHOR]

Novel naphthoquinones were designed,synthesized, and tested assubstrate-based inhibitors against the membrane-embedded protein quinol/fumaratereductase (QFR) from Wolinella succinogenes, a targetclosely related to QFRs from the human pathogens Helicobacterpyloriand Campylobacter jejuni. For a betterunderstanding of the hitherto structurally unexplored substrate bindingpocket, a structure–activity relationship (SAR) study was carriedout. Analogues of lawsone (2-hydroxy-1,4-naphthoquinone 3a) were synthesized that vary in length and size of the alkyl sidechains (3b–k). A combined study onthe prototropic tautomerism of 2-hydroxy-1,4-naphthoquinones seriesindicated that the 1,4-tautomer is the more stable and biologicallyrelevant isomer and that the presence of the hydroxyl group is crucialfor inhibition. Furthermore, 2-bromine-1,4-naphthoquinone (4a–c) and 2-methoxy-1,4-naphthoquinone (5a–b) series were also discovered as novel andpotent inhibitors. Compounds 4aand 4bshowedIC50values in low micromolar range in the primary assayand no activity in the counter DT-diaphorase assay. [ABSTRACT FROM AUTHOR]

In Gram-positive bacteria and other prokaryotes containing succinate:menaquinone reductases, it has previously been shown that the succinate oxidase and succinate:menaquinone reductase activities are lost when the transmembrane electrochemical proton potential, Δp, is abolished by the rupture of the bacteria or by the addition of a protonophore. It has been proposed that the endergonic reduction of menaquinone by succinate is driven by the electrochemical proton potential. Opposite sides of the cytoplasmic membrane were envisaged to be separately involved in the binding of protons upon the reduction of menaquinone and their release upon succinate oxidation, with the two reactions linked by the transfer of two electrons through the enzyme. However, it has previously been argued that the observed Δp dependence is not associated specifically with the succinate:menaquinone reductase. Definitive insight into the mechanism of catalysis of this reaction requires a corresponding functional characterization of an isolated, membrane-bound succinate:menaquinone reductase from a Gram-positive bacterium. Here, we describe the purification, reconstitution into proteoliposomes, and functional characterization of the diheme-containing succinate:menaquinone reductase from the Gram-positive bacterium Bacillus licheniformis and, with the help of the design, synthesis, and characterization of quinones with finely tuned oxidation/reduction potentials, provide unequivocal evidence for Δp-dependent catalysis of succinate oxidation by quinone as well as for Δp generation upon catalysis of fumarate reduction by quinol. [ABSTRACT FROM AUTHOR]

Reconciliation of apparently contradictory experimental results obtained on the quinol:fumarate reductase, a diheme-containing respiratory membrane protein complex from Wolinella succino- genes, was previously obtained by the proposal of the so-called "E pathway hypothesis." According to this hypothesis, transmembrane electron transfer via the heme groups is strictly coupled to cotransfer of protons via a transiently established pathway thought to contain the side chain of residue Glu-C180 as the most prominent component. Here we demonstrate that, after replacement of Glu-C180 with Gln or lle by site-directed mutagenesis, the resulting mutants are unable to grow on fumarate, and the membrane-bound variant enzymes lack quinol oxidation activity. Upon solubilization, however, the purified enzymes display ≈1/10 of the specific quinol oxidation activity of the wild-type enzyme and unchanged quinol Michaelis constants, Km. The refined x-ray crystal structures at 2.19 Å and 2.76 Å resolution, respectively, rule out major structural changes to account for these experimental observations. Changes in the oxidation-reduction heme midpoint potential allow the conclusion that deprotonation of Glu-C180 in the wild-type enzyme facilitates the reoxidation of the reduced high-potential heme. Comparison of solvent isotope effects indicates that a rate-limiting proton transfer step in the wild-type enzyme is lost in the Glu-C180 → Gln variant. The results provide experimental evidence for the validity of the E pathway hypothesis and for a crucial functional role of Glu-C180. [ABSTRACT FROM AUTHOR]

The title compound, C14H12O4, forms crystals which appear monoclinic but are actually twinned triclinic. The asymmetric unit consists of two similar mol­ecules, which differ only in the conformation of the 3-oxobutyl side chain. The mol­ecular conformation is characterized by an intra­molecular O—H⋯O hydrogen bond between the hydroxy group and the adjacent carbonyl O atom. The crystal structure is stabilized by O—H⋯O hydrogen bonds connecting the mol­ecules into zigzag chains running along the b axis. [ABSTRACT FROM AUTHOR]

The Staphylococcus epidermidis glucose/H1 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/H1 symport, respectively. Unlike other bacterial GLUT homologs (for example, XylE), GlcPSe has a loose H1/sugar coupling. Asp22 is part of the proton-binding site of GlcPSe and crucial for the glucose/H1 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 6 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/H1 symport. [ABSTRACT FROM AUTHOR]

Polycystin-2 (PC2), a calcium-activated cation TRP channel, is involved in diverse Ca 2+ signaling pathways. Malfunctioning Ca 2+ 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, PC2 SI ), whereas multiple cations are bound along the translocation pathway in the second structure (multi-ion mode, PC2 MI ). Ca 2+ binding at the entrance of the selectivity filter suggests Ca 2+ blockage in PC2 MI , and we observed density for the Ca 2+ -sensing C-terminal EF hand in the unblocked PC2 SI 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.