Opening and closing of the periplasmic gate in lactose permease

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Few side chains in the galactoside/H + symporter LacY (lactose permease of Escherichia coli) are irreplaceable for an alternating access mechanism in which sugar binding induces closing of the cytoplasmic cavity and reciprocal opening of a periplasmic cavity. In this study, each irreplaceable residue was mutated individually, and galactoside-induced opening or closing of periplasmic or cytoplasmic cavities was probed by site-directed alkylation. Mutation of Glu126 (helix IV) or Arg144 (helix V), which are essential for sugar binding, completely blocks sugar-induced periplasmic opening as expected. Remarkably, however, replacement of Glu269 (helix VIII), His322 (helix X), or Tyr236 (helix VII) causes spontaneous opening of the periplasmic cavity in the absence of sugar and decreased closing of the cytoplasmic cavity in the presence of galactoside. In contrast, mutation of Arg302 (helix IX) or Glu325 (helix X) has no such effect, and sugar binding induces normal opening and closing of periplasmic and cytoplasmic cavities. Possibly, Glu269, His322, and Tyr236 act in concert to coordinate opening and closing of the cavities by binding water, which also acts as a cofactor in H + translocation. Mutation of the triad results in loss of the bound water, which destabilizes LacY, and the cavities open and close in an uncoordinated manner. Thus, the triad mutants exhibit poor affinity for sugar, and galactoside/H + symport is abolished as well.membrane protein | membranes | protein dynamics | transport T he lactose permease of Escherichia coli (LacY), a member of the major facilitator superfamily of membrane transport proteins, catalyzes the coupled translocation of a galactoside and an H + (i.e., galactoside/H + symport). Thus, LacY transduces free energy stored in an H + electrochemical gradient (Δμ H +) into a galactoside concentration gradient. As transport is obligatorily coupled, LacY also transduces free energy stored in a sugar concentration gradient into Δμ H +, the polarity of which depends on the direction of the sugar gradient (reviewed in ref. 1). LacY has been solubilized from the membrane, purified to homogeneity in a highly functional state (2), and is structurally (3, 4), as well as functionally (5), a monomer.X-ray crystal structures of LacY (6-9) and various independent biochemical and spectroscopic findings (10-17) provide converging evidence for an alternating access mechanism. By this means, sugar binding induces coordinated opening and closing of periplasmic and cytoplasmic cavities, respectively, thereby allowing accessibility of sugar-and H + -binding sites to either side of the membrane (i.e., the alternating access model; reviewed in refs. 18, 19). A similar model has been proposed for a number of other transporters (e.g., refs. 20-24). It is also likely that the alternating access model for LacY involves formation of an occluded intermediate (25,26), which is consistent with the highly dynamic nature of the protein (15, 27-31).Cys-scanning and site-directed mutagenesis of each residue in LacY de...

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confidence: 64%

Role of the irreplaceable residues in the LacY alternating access mechanism

Zhou¹,

Jiang²,

Kaback

2012

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“…A similar model has been proposed for the glycerol phosphate/phosphate antiporter GlpT, a related MFS protein (17) and the ABC transporter Sav 1866 (18). The alternating-access model involves a global conformational change, which is consistent with the highly dynamic nature of LacY (7,13,14,16,(19)(20)(21)(22)(23).A simple kinetic model for lactose/H ϩ symport (Fig. 1) has been proposed based on extensive studies of partial reactions (efflux, equilibrium exchange, and entrance counterflow) catalyzed by LacY and site-directed mutants defective in the symport mechanism (see ref.…”

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confidence: 67%

“…X-ray crystal structures of LacY (8)(9)(10), all of which are in an inward-facing conformation, and a wealth of biochemical/ biophysical data (7,(11)(12)(13)(14)(15)(16) provide evidence for an alternatingaccess mechanism of action. By this means, upon sugar binding or imposition of a ⌬ Hϩ ) (interior negative and/or alkaline), the inward-facing cavity closes with opening of an outward-facing cavity, thereby allowing alternative accessibility of the sugarbinding site, which is at the apex of the cavity in the middle of the molecule, to either side of the membrane.…”

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confidence: 99%

Protonation and sugar binding to LacY

Смирнова

Kasho²,

Kaback³

2008

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The effect of bulk-phase pH on the apparent affinity (K d app ) of purified wild-type lactose permease (LacY) for sugars was studied. K d app values were determined by ligand-induced changes in the fluorescence of either of two covalently bound fluorescent reporters positioned away from the sugar-binding site. K d app for three different galactopyranosides was determined over a pH range from 5.5 to 11. A remarkably high pK a of Ϸ10.5 was obtained for all sugars. Kinetic data for thiodigalactoside binding measured from pH 6 to 10 show that decreased affinity for sugar at alkaline pH is due specifically to increased reverse rate. A similar effect was also observed with nitrophenylgalactoside by using a direct binding assay. Because affinity for sugar remains constant from pH 5.5 to pH 9.0, it follows that LacY is fully protonated with respect to sugar binding under physiological conditions of pH. The results are consistent with the conclusion that LacY is protonated before sugar binding during lactose/H ؉ symport in either direction across the membrane.lactose permease ͉ membrane transporters ͉ pH titrations ͉ proton translocation ͉ substrate affinity T he lactose permease of Escherichia coli (LacY), a paradigm for the major facilitator superfamily (MFS) of membrane transport proteins (1, 2), translocates a galactosidic sugar and an H ϩ across the cytoplasmic membrane (3, 4). This coupled translocation mechanism enables LacY to use free energy stored in an electrochemical H ϩ gradient (⌬ Hϩ ) to drive sugar accumulation against a concentration gradient (5-7).X-ray crystal structures of LacY (8-10), all of which are in an inward-facing conformation, and a wealth of biochemical/ biophysical data (7, 11-16) provide evidence for an alternatingaccess mechanism of action. By this means, upon sugar binding or imposition of a ⌬ Hϩ ) (interior negative and/or alkaline), the inward-facing cavity closes with opening of an outward-facing cavity, thereby allowing alternative accessibility of the sugarbinding site, which is at the apex of the cavity in the middle of the molecule, to either side of the membrane. A similar model has been proposed for the glycerol phosphate/phosphate antiporter GlpT, a related MFS protein (17) and the ABC transporter Sav 1866 (18). The alternating-access model involves a global conformational change, which is consistent with the highly dynamic nature of LacY (7,13,14,16,(19)(20)(21)(22)(23).A simple kinetic model for lactose/H ϩ symport (Fig. 1) has been proposed based on extensive studies of partial reactions (efflux, equilibrium exchange, and entrance counterflow) catalyzed by LacY and site-directed mutants defective in the symport mechanism (see ref. 5). Wild-type LacY is tightly coupled with respect to sugar and H ϩ translocation and does not translocate H ϩ without substrate or vice versa. The effect of ambient pH on ⌬ Hϩ -driven symport is bell-shaped with an optimum at approximately pH 7.5 (24, 25), whereas the partial reactions exhibit different dependences on pH. For example, rates of lac...

“…Thus, measurement of interspin distances with nitroxide-labeled Cys pairs in LacY reveals that sugar binding induces a decrease in distances on the cytoplasmic side and a corresponding increase in distances on the periplasmic side (13,14). Site-directed alkylation of single Cys LacY mutants in either right-side-out membrane vesicles (15,16) or dodecyl-β-D-maltopyranoside (DDM) micelles (10), as well as single-molecule fluorescence (17) and thiol cross-linking (18), also indicates that sugar binding increases the probability of opening on the periplasmic side and closing on the cytoplasmic side.…”

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confidence: 99%

Real-time conformational changes in LacY

Смирнова¹,

Kasho²,

Kaback

2014

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Galactoside/H + symport across the cytoplasmic membrane of Escherichia coli is catalyzed by lactose permease (LacY), which uses an alternating access mechanism with opening and closing of deep cavities on the periplasmic and cytoplasmic sides. In this study, conformational changes in LacY initiated by galactoside binding were monitored in real time by Trp quenching/unquenching of bimane, a small fluorophore covalently attached to the protein.Rates of change in bimane fluorescence on either side of LacY were measured by stopped flow with LacY in detergent or in proteoliposomes and were compared with rates of galactoside binding. With LacY in proteoliposomes, the periplasmic cavity is tightly sealed and the substrate-binding rate is limited by the rate of opening of this cavity. Rates of opening, measured as unquenching of bimane fluorescence, are 20-30 s −1 , independent of sugar concentration and essentially the same in detergent or in proteoliposomes. On the cytoplasmic side of LacY in proteoliposomes, slow bimane quenching (i.e., closing of the cavity) is observed at a rate that is also independent of sugar concentration and similar to the rate of sugar binding from the periplasmic side. Therefore, opening of the periplasmic cavity not only limits access of sugar to the binding site of LacY but also controls the rate of closing of the cytoplasmic cavity. LacY is organized into two pseudosymmetrical bundles, each containing six transmembrane helices, most of which are irregular, with the N and C termini on the cytoplasmic side of the membrane. WT LacY and a conformationally restricted mutant exhibit an inward-facing conformation with a tightly sealed periplasmic side and a water-filled cavity open to the cytoplasm (6-9), which is the conformation present in the membrane in the absence of sugar (10). Another conformation has been observed recently with a double-Trp mutant (11) that exhibits an occluded galactoside molecule with a narrow opening on the periplasmic side and a tightly sealed cytoplasmic aspect (12) (Fig. 1A).LacY is highly dynamic and exhibits multiple conformations at any given time, and galactoside binding triggers a shift between conformers (1). Thus, measurement of interspin distances with nitroxide-labeled Cys pairs in LacY reveals that sugar binding induces a decrease in distances on the cytoplasmic side and a corresponding increase in distances on the periplasmic side (13,14). Site-directed alkylation of single Cys LacY mutants in either right-side-out membrane vesicles (15, 16) or dodecyl-β-D-maltopyranoside (DDM) micelles (10), as well as single-molecule fluorescence (17) and thiol cross-linking (18), also indicates that sugar binding increases the probability of opening on the periplasmic side and closing on the cytoplasmic side.Recently, conformational changes in LacY have been studied dynamically by site-directed Trp fluorescence quenching by a protonated His or Lys residue (19). Sugar binding leads to unquenching of Trp fluorescence in periplasmic LacY mutants N245W (helix VII) or F378...