Apo-intermediate in the transport cycle of lactose permease (LacY).
ABSTRACT: The lactose permease (LacY) catalyzes coupled stoichiometric symport of a galactoside and an H(+). Crystal structures reveal 12, mostly irregular, transmembrane ?-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.
Project description:The lactose permease of Escherichia coli (LacY) utilizes an alternating access symport mechanism with multiple conformational intermediates, but only inward (cytoplasmic)- or outward (periplasmic)-open structures have been characterized by X-ray crystallography. It is demonstrated here with sugar-binding studies that cross-linking paired-Cys replacements across the closed cytoplasmic cavity stabilize an occluded conformer with an inaccessible sugar-binding site. In addition, a nanobody (Nb) that stabilizes a periplasmic-open conformer with an easily accessible sugar-binding site in WT LacY fails to cause the cytoplasmic cross-linked mutants to become accessible to galactoside, showing that the periplasmic cavity is closed. These results are consistent with tight association of the periplasmic ends in two pairs of helices containing clusters of small residues in the packing interface between N- and C-terminal six-helix bundles of the symporter. However, after reduction of the disulfide bond, the Nb markedly increases the rate of galactoside binding, indicating unrestricted access to the Nb epitope and the galactoside-binding site from the periplasm. The findings indicate that the cross-linked cytoplasmic double-Cys mutants resemble an occluded apo-intermediate in the transport cycle.
Project description:According to x-ray structure, the lactose permease (LacY) is a monomer organized into N- and C-terminal six-helix bundles that form a deep internal cavity open on the cytoplasmic side with a single sugar-binding site at the apex. The periplasmic side of the molecule is closed. During sugar/H(+) symport, a cavity facing the periplasmic side is thought to open with closure of the inward-facing cytoplasmic cavity so that the sugar-binding site is alternately accessible to either face of the membrane. Double electron-electron resonance (DEER) is used here to measure interhelical distance changes induced by sugar binding to LacY. Nitroxide-labeled paired-Cys replacements were constructed at the ends of transmembrane helices on the cytoplasmic or periplasmic sides of wild-type LacY and in the conformationally restricted mutant Cys-154-->Gly. Distances were then determined in the presence of galactosidic or nongalactosidic sugars. Strikingly, specific binding causes conformational rearrangement on both sides of the molecule. On the cytoplasmic side, each of six nitroxide-labeled pairs exhibits decreased interspin distances ranging from 4 to 21 A. Conversely, on the periplasmic side, each of three spin-labeled pairs shows increased distances ranging from 4 to 14 A. Thus, the inward-facing cytoplasmic cavity closes, and a cleft opens on the tightly packed periplasmic side. In the Cys-154-->Gly mutant, sugar-induced closing is observed on the cytoplasmic face, but little or no change occurs on periplasmic side. The DEER measurements in conjunction with molecular modeling based on the x-ray structure provide strong support for the alternative access model and reveal a structure for the outward-facing conformer of LacY.
Project description:Lactose permease of Escherichia coli (LacY) is highly dynamic, and sugar binding causes closing of a large inward-facing cavity with opening of a wide outward-facing hydrophilic cavity. Therefore, lactose/H(+) symport via LacY very likely involves a global conformational change that allows alternating access of single sugar- and H(+)-binding sites to either side of the membrane. Here, in honor of Stephan H. White's seventieth birthday, we review in camera the various biochemical/biophysical approaches that provide experimental evidence for the alternating access mechanism.
Project description:Galactoside/H+ symport by the lactose permease of Escherichia coli (LacY) involves reciprocal opening and closing of periplasmic and cytoplasmic cavities so that sugar- and H+-binding sites become alternatively accessible to either side of the membrane. After reconstitution into proteoliposomes, LacY with the periplasmic cavity sealed by cross-linking paired-Cys residues does not bind sugar from the periplasmic side. However, reduction of the S-S bond restores opening of the periplasmic cavity and galactoside binding. Furthermore, nanobodies that stabilize the double-Cys mutant in a periplasmic-open conformation and allow free access of galactoside to the binding site do so only after reduction of the S-S bond. In contrast, when cross-linked LacY is solubilized in detergent, galactoside binding is observed, indicating that the cytoplasmic cavity is patent. Sugar binding from the cytoplasmic side exhibits nonlinear stopped-flow kinetics, and analysis reveals a two-step process in which a conformational change precedes binding. Because the cytoplasmic cavity is spontaneously closing and opening in the symporter with a sealed periplasmic cavity, it is apparent that an asymmetrical conformational transition controls access of sugar to the binding site.
Project description: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 pK(a) of 8, typical for a His side chain interacting with an aromatic group. As estimated from stopped-flow studies, the rate of sugar-induced opening is approximately 100 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-140-->Trp/Phe-334-->His exhibits sugar-induced Trp quenching. Again, quenching is pH dependent (pK(a) = 8), but remarkably, the rate of sugar-induced quenching is only approximately 0.4 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.
Project description:Many independent lines of evidence indicate that the lactose permease of Escherichia coli (LacY) is highly dynamic and that sugar binding causes closing of a large inward-facing cavity with opening of a wide outward-facing hydrophilic cavity. Therefore, lactose/H(+) symport catalyzed by LacY very likely involves a global conformational change that allows alternating access of single sugar- and H(+)-binding sites to either side of the membrane (the alternating access model). The x-ray crystal structures of LacY, as well as the majority of spectroscopic studies, use purified protein in detergent micelles. By using site-directed alkylation, we now demonstrate that sugar binding induces virtually the same global conformational change in LacY whether the protein is in the native bacterial membrane or is solubilized and purified in detergent. The results also indicate that the x-ray crystal structure reflects the structure of wild-type LacY in the native membrane in the absence of sugar.
Project description:The N- and C-terminal six-helix bundles of lactose permease (LacY) form a large internal cavity open on the cytoplasmic side and closed on the periplasmic side with a single sugar-binding site at the apex of the cavity near the middle of the molecule. During sugar/H(+) symport, an outward-facing cavity is thought to open with closing of the inward-facing cavity so that the sugar-binding site is alternately accessible to either face of the membrane. In this communication, single-molecule fluorescence (Förster) resonance energy transfer is used to test this model with wild-type LacY and a conformationally restricted mutant. Pairs of Cys residues at the ends of two helices on the cytoplasmic or periplasmic sides of wild-type LacY and the mutant were labeled with appropriate donor and acceptor fluorophores, single-molecule fluorescence resonance energy transfer was determined in the absence and presence of sugar, and distance changes were calculated. With wild-type LacY, binding of a galactopyranoside, but not a glucopyranoside, results in a decrease in distance on the cytoplasmic side and an increase in distance on the periplasmic side. In contrast, with the mutant, a more pronounced decrease in distance and in distance distribution is observed on the cytoplasmic side, but there is no change on the periplasmic side. The results are consistent with the alternating access model and indicate that the defect in the mutant is due to impaired ligand-induced flexibility on the periplasmic side.
Project description:Lactose permease of Escherichia coli (LacY) catalyzes symport of a galactopyranoside and an H? via an alternating access mechanism. The transition from an inward- to an outward-facing conformation of LacY involves sugar-release followed by deprotonation. Because the transition depends intimately upon the dynamics of LacY in a bilayer environment, molecular dynamics (MD) simulations may be the only means of following the accompanying structural changes in atomic detail. Here, we describe MD simulations of wild-type apo LacY in phosphatidylethanolamine (POPE) lipids that features two protonation states of the critical Glu325. While the protonated system displays configurational stability, deprotonation of Glu325 causes significant structural rearrangements that bring into proximity side chains important for H? translocation and sugar binding and closes the internal cavity. Moreover, protonated LacY in phosphatidylcholine (DMPC) lipids shows that the observed dynamics are lipid-dependent. Together, the simulations describe early dynamics of the inward-to-outward transition of LacY that agree well with experimental data.
Project description:The lactose permease (LacY) of Escherichia coli is the prototype of the major facilitator superfamily, one of the largest families of membrane transport proteins. Structurally, two pseudo-symmetrical six-helix bundles surround a large internal aqueous cavity. Single binding sites for galactoside and H+ are positioned at the approximate center of LacY halfway through the membrane at the apex of the internal cavity. These features enable LacY to function by an alternating-access mechanism that can catalyze galactoside/H+ symport in either direction across the cytoplasmic membrane. The H+-binding site is fully protonated under physiological conditions, and subsequent sugar binding causes transition of the ternary complex to an occluded intermediate that can open to either side of the membrane. We review the structural and functional evidence that has provided new insight into the mechanism by which LacY achieves active transport against a concentration gradient.
Project description:X-ray crystal structures of LacY (lactose permease of Escherichia coli) exhibit a large cytoplasmic cavity containing the residues involved in sugar binding and H(+) translocation at the apex and a tightly packed side facing the periplasm. However, biochemical and biophysical evidence provide a strong indication that a hydrophilic pathway opens on the external surface of LacY with closing of the cytoplasmic side upon sugar binding. Thus, an alternating-access mechanism in which sugar- and H(+)-binding sites at the approximate middle of the molecule are alternatively exposed to either side of the membrane is likely to underlie LacY-catalyzed sugar/H(+) symport. To further investigate periplasmic opening, we replaced paired residues on the tightly packed periplasmic side of LacY with Cys, and the effect of cross-linking was studied by testing the accessibility/reactivity of Cys148 with the elongated ( approximately 29 A), impermeant hydrophilic reagent maleimide-PEG2-biotin. When the paired-Cys mutant Ile40-->Cys/Asn245-->Cys containing native Cys148 is oxidized to form a disulfide bond, the reactivity of Cys148 is markedly inhibited. Moreover, the reactivity of Cys148 in this mutant increases with the length of the cross-linking agent. In contrast, maleimide-PEG2-biotin reactivity of Cys148 is unaffected by oxidation of two other paired-Cys mutants at the mouth of the periplasmic cavity. The data indicate that residues Ile40 and Asn245 play a primary role in gating the periplasmic cavity and provide further support for the alternating-access model.