Electrophysiological characterization of LacY

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Sugar/H ؉ symport by lactose permease (LacY) utilizes an alternating access mechanism in which sugar and H ؉ binding sites in the middle of the molecule are alternatively exposed to either side of the membrane by sequential opening and closing of inward-and outward-facing hydrophilic cavities. Here, we introduce Trp residues on either side of LacY where they are predicted to be in close proximity to side chains of natural Trp quenchers in either the inward-or outward-facing conformers. In the inward-facing conformer, LacY is tightly packed on the periplasmic side, and Trp residues placed at positions 245 (helix VII) or 378 (helix XII) are in close contact with His-35 (helix I) or Lys-42 (helix II), respectively. Sugar binding leads to unquenching of Trp fluorescence in both mutants, a finding clearly consistent with opening of the periplasmic cavity. The pH dependence of Trp-245 unquenching exhibits a pKa of 8, typical for a His side chain interacting with an aromatic group. As estimated from stopped-flow studies, the rate of sugarinduced opening is Ϸ100 s ؊1 . On the cytoplasmic side, Phe-140 (helix V) and Phe-334 (helix X) are located on opposite sides of a wide-open hydrophilic cavity. In precisely the opposite fashion from the periplasmic side, mutant Phe-1403 Trp/Phe-3343 His exhibits sugar-induced Trp quenching. Again, quenching is pH dependent (pKa ‫؍‬ 8), but remarkably, the rate of sugar-induced quenching is only Ϸ0.4 s ؊1 . The results provide yet another strong, independent line of evidence for the alternating access mechanism and demonstrate that the methodology described provides a sensitive probe to measure rates of conformational change in membrane transport proteins.lactose permease ͉ membrane transporters ͉ site-directed mutagenesis ͉ stopped-flow ͉ tryptophan fluorescence T he lactose permease of Escherichia coli (LacY) is a galactopyranoside/H ϩ symporter that belongs to the Major Facilitator Superfamily (MFS) of membrane transport proteins (1, 2). LacY is arguably the most extensively studied, ion-coupled, membrane transport protein (3-7). Several X-ray structures (8-10) reveal an inward-facing conformation with a tightly packed, closed periplasmic side, and a large hydrophilic cavity open to the cytoplasm with sugar-and H ϩ -binding sites in the middle of the molecule. Although an outward-open conformation has not yet been obtained crystallographically, it is well-documented that LacY undergoes conformational changes upon sugar binding that lead to closing of the cytoplasmic cavity and opening of a relatively large hydrophilic periplasmic cavity. Thus, underestimates of cross-linking distances in the inward-facing structure (11), site-directed alkylation (12-14), single-molecule Förster resonance energy transfer (15), double electron-electron resonance (16), and site-directed thiol crosslinking (17) all provide independent evidence that sugar binding induces an outward-facing conformation with a large periplasmic opening and closure of the inward-facing cavity. However, steadystate measureme...

Section: Discussionmentioning

confidence: 94%

Probing of the rates of alternating access in LacY with Trp fluorescence

Смирнова¹,

Kasho²,

Sugihara³

et al. 2009

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“…8 A and B, dark blue, steps [5][6][7][8], transport proceeds at the same rate in both directions. Therefore, the activation energy barriers must be equal from both sides (Fig.…”

Section: Discussionmentioning

confidence: 99%

“…8 A and B, opening of empty LacY to the periplasm, steps 1-4) and from outward-facing to inward-facing following the blue lines (Fig. 8 A and B, transport of sugar from periplasm to cytoplasm, steps [5][6][7][8]. High occurrence signifies a lowenergy state [ Fig.…”

Section: Discussionmentioning

confidence: 99%

“…MFS proteins catalyze transport of a wide range of substrates, including amines, acids, amino acids, sugars, peptides, and antibiotics, in many instances, by transducing the energy stored in an H + electrochemical gradient into a concentration gradient of substrate. The lactose permease of Escherichia coli (LacY), which catalyzes the coupled transport of a galactopyranoside and an H + (galactoside/H + symport) (3)(4)(5)(6), is arguably the most extensively studied member of the MFS (7,8). Essentially all 417 amino acyl side chains in LacY have been mutagenized (9), and functional analyses of the mutants reveals that fewer than 10 side chains play a central role in the symport mechanism ( Fig.…”

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confidence: 99%

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

Madej¹,

Soro²,

Kaback

2012

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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. proteins represents the largest family of secondary transporters, with members from Archaea to Homo sapiens (1, 2). MFS proteins catalyze transport of a wide range of substrates, including amines, acids, amino acids, sugars, peptides, and antibiotics, in many instances, by transducing the energy stored in an H + electrochemical gradient into a concentration gradient of substrate. The lactose permease of Escherichia coli (LacY), which catalyzes the coupled transport of a galactopyranoside and an H + (galactoside/H + symport) (3-6), is arguably the most extensively studied member of the MFS (7,8). Essentially all 417 amino acyl side chains in LacY have been mutagenized (9), and functional analyses of the mutants reveals that fewer than 10 side chains play a central role in the symport mechanism ( Fig. 1 A and B): Glu126 (helix IV), Arg144 (helix V), and Trp151 (helix V) are directly involved in galactoside recognition and binding; Tyr236 (helix VII), Glu269 (helix VIII), and His322 (helix X) are involved in both H + translocation and affinity for sugar; and Arg302 (helix IX) and Glu325 (helix X) play important roles in H + translocation (7, 10). In the available inward-facing crystal structures of , these residues are located at the apex of a deep central hydrophilic cavity, which is open to the cytoplasm only ( Fig. 1 A and B). The cavity is formed by 12 mostly irregular transmembrane helices organized in two pseudosymmetrical 6-helix bundles (helices I-VI and helices VII-XII) (11)(12)(13)(14).A six-step kinetic model for lactose/H + symport has been proposed (15, 16) (Fig. 1C). The steps include (i) deprotonation of LacY, (ii) a conformational change allowing the galactoside-and H + -binding sites to become accessible to the other side of the membrane, (iii) protonation of LacY, (iv) substrate binding, (v) a global conformational change in which the protonated LacY with bound sugar returns to the initial inward-facing conformation, and (vi) dissociation of sugar. This seque...

“…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)(4)(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.…”

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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...