Crystal structure of lactose permease in complex with an affinity inactivator yields unique insight into sugar recognition

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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...

Section: Discussionmentioning

confidence: 99%

Role of the irreplaceable residues in the LacY alternating access mechanism

Zhou¹,

Jiang²,

Kaback

2012

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“…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)(7)(8)(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.…”

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confidence: 92%

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...

“…6). Nevertheless, all X-ray structures of LacY obtained so far exhibit an inward-facing conformation, with the sugar-binding site at the apex of a deep hydrophilic cavity open to the cytoplasmic side only (7)(8)(9)(10). In this conformation, the periplasmic side is tightly sealed and the sugar-binding site is inaccessible from the periplasm.…”

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confidence: 99%

Trp replacements for tightly interacting Gly–Gly pairs in LacY stabilize an outward-facing conformation

Смирнова¹,

Kasho²,

Sugihara³

et al. 2013

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Trp replacements for conserved Gly-Gly pairs between the N-and C-terminal six-helix bundles on the periplasmic side of lactose permease (LacY) cause complete loss of transport activity with little or no effect on sugar binding. Moreover, the detergent-solubilized mutants exhibit much greater thermal stability than WT LacY. A Cys replacement for Asn245, which is inaccessible/unreactive in WT LacY, alkylates readily in the Gly→Trp mutants, indicating that the periplasmic cavity is patent. Stopped-flow kinetic measurements of sugar binding with the Gly→Trp mutants in detergent reveal linear dependence of binding rates on sugar concentration, as observed with WT or the C154G mutant of LacY, and are compatible with free access to the sugar-binding site in the middle of the molecule. Remarkably, after reconstitution of the Gly→Trp mutants into proteoliposomes, the concentration dependence of sugar-binding rates increases sharply with even faster rates than measured in detergent. Such behavior is strikingly different from that observed for reconstituted WT LacY, in which sugar-binding rates are independent of sugar concentration because opening of the periplasmic cavity is limiting for sugar binding. The observations clearly indicate that Gly→Trp replacements, which introduce bulky residues into tight Gly-Gly interdomain interactions on the periplasmic side of LacY, prevent closure of the periplasmic cavity and, as a result, shift the distribution of LacY toward an outwardopen conformation.alternating access | fluorescence | major facilitator superfamily | permease | symport T he lactose permease (LacY) of Escherichia coli, the most intensively studied member of the major facilitator superfamily (1, 2), catalyzes coupled, stoichiometric translocation of a galactopyranoside and an H + (sugar/H + symport) across the cytoplasmic membrane (3-5). Because sugar and H + translocation are obligatorily coupled, LacY transduces the free energy stored in an H + electrochemical gradient (Δμ̃H+) into a sugar concentration gradient; conversely, downhill transport of galactoside drives uphill transport of H + with the generation of Δμ̃H+, the polarity of which is dictated by the direction of the sugar concentration gradient. The highly dynamic nature of LacY is consistent with an alternating access mechanism involving a global conformational change in which sugar and H + binding sites are alternatively exposed to either side of the membrane (reviewed in ref. 6). Nevertheless, all X-ray structures of LacY obtained so far exhibit an inward-facing conformation, with the sugar-binding site at the apex of a deep hydrophilic cavity open to the cytoplasmic side only (7-10). In this conformation, the periplasmic side is tightly sealed and the sugar-binding site is inaccessible from the periplasm. However, a wealth of biophysical and spectroscopic data, which include site-directed alkylation (11-16), single-molecule fluorescence resonance energy transfer (FRET) (17), double electron-electron resonance (18), site-directed cross-linking (19) an...