Structural determination of wild-type lactose permease

Department of Physiology, University of California, Los Angeles, CA 90095-1662, USA.
Proceedings of the National Academy of Sciences (Impact Factor: 9.81). 10/2007; 104(39):15294-8. DOI: 10.1073/pnas.0707688104
Source: PubMed

ABSTRACT Here we describe an x-ray structure of wild-type lactose permease (LacY) from Escherichia coli determined by manipulating phospholipid content during crystallization. The structure exhibits the same global fold as the previous x-ray structures of a mutant that binds sugar but cannot catalyze translocation across the membrane. LacY is organized into two six-helix bundles with twofold pseudosymmetry separated by a large interior hydrophilic cavity open only to the cytoplasmic side and containing the side chains important for sugar and H(+) binding. To initiate transport, binding of sugar and/or an H(+) electrochemical gradient increases the probability of opening on the periplasmic side. Because the inward-facing conformation represents the lowest free-energy state, the rate-limiting step for transport may be the conformational change leading to the outward-facing conformation.

Download full-text


Available from: Osman Mirza, Jul 28, 2015
  • Source
    • "The lactose permease of Escherichia coli (LacY) with 12 mostly irregular transmembrane a helices organized into two pseudosymmetrical six-helix bundles connected by a relatively long cytoplasmic loop (Abramson et al., 2003; Chaptal et al., 2011; Guan et al., 2007; Kumar et al., 2014; Mirza et al., 2006) is a paradigm for the major facilitator superfamily (MFS) (Marger and Saier, 1993). In the native membrane, which is 70%–80% zwitterionic phosphatidylethanolamine (PE) and 20%–25% anionic phosphatidylglycerol (PG) plus cardiolipin, LacY adopts a native topology with the N and C termini on the cytoplasmic surface of the membrane (Bogdanov et al., 2008; Calamia and Manoil, 1990; Chen and Wilson, 1984; Foster et al., 1983; Seto-Young et al., 1985). "
    [Show abstract] [Hide abstract]
    ABSTRACT: Lipids of the Escherichia coli membrane are mainly composed of 70%-80% phosphatidylethanolamine (PE) and 20%-25% phosphatidylglycerol (PG). Biochemical studies indicate that the depletion of PE causes inversion of the N-terminal helix bundle of the lactose permease (LacY), and helix VII becomes extramembranous. Here we study this phenomenon using single-molecule force spectroscopy, which is sensitive to the structure of membrane proteins. In PE and PG at a ratio of 3:1, ∼95% of the LacY molecules adopt a native structure. However, when PE is omitted and the membrane contains PG only, LacY almost equally populates a native and a perturbed conformation. The most drastic changes occur at helices VI and VII and the intervening loop. Since helix VII contains Asp237 and Asp240, zwitterionic PE may suppress electrostatic repulsion between LacY and PG in the PE:PG environment. Thus, PE promotes a native fold and prevents LacY from populating a functionally defective, nonnative conformation. Copyright © 2015 Elsevier Ltd. All rights reserved.
    Structure 03/2015; 23(4). DOI:10.1016/j.str.2015.02.009 · 6.79 Impact Factor
  • Source
    • "Following years of effort, the first three-dimensional structure of LacY was resolved from X-ray diffraction studies using the conformationally restricted mutant (C154G; Cys 154 ! Gly) (Abramson et al., 2003) and, shortly after, the wild-type structure was also completed (Guan et al., 2007). In side view, the monomer of LacY is heart-shaped, and accounts for a diameter of 6 nm displaying a large internal hydrophobic cavity open to the cytoplasmic side. "
    [Show abstract] [Hide abstract]
    ABSTRACT: AbstractFörster resonance energy transfer (FRET) is a photophysical process by which a donor (D) molecule in an electronic excited state transfers its excitation energy to a second species, the acceptor (A). Since FRET efficiency depends on D-A separation, the measurement of donor fluorescence in presence and absence of the acceptor allows determination of this distance, and therefore FRET has been extensively used as a “spectroscopic ruler”. In membranes, interpretation of FRET is more complex, since one D may be surrounded by many A molecules. Such is the case encountered with membrane proteins and lipids in the bilayer. This paper reviews the application of a model built to analyze FRET data between a single tryptophan mutant of the transmembrane protein lactose permease (W151/C154G of LacY), the sugar/H+ symporter from Escherichia coli, and different pyrene-labeled phospholipids. Several variables of the system with biological implication have been investigated: The selectivity of LacY for different species of phospholipids, the enhancement of the sensitivity of the FRET modeling, and the mutation of a particular aminoacid (D68C) of the protein. The results obtained support: (i) Preference of LacY for phosphatidylethanolamine (PE) over phosphatidylglycerol (PG); (ii) affinity of LacY for fluid (L α) phases; and (iii) importance of the aspartic acid in position 68 in the sequence of LacY regarding the interaction with the phospholipid environment. Besides, by exploring the enhancement of the sensitivity by using pure lipid matrices with higher mole fractions of labelled-phospholipid, the dependence on acyl chain composition is unveiled.
    Molecular Membrane Biology 06/2014; 31(4). DOI:10.3109/09687688.2014.915351 · 1.73 Impact Factor
  • Source
    • "Examination of the proposal of Madej et al. [2013] was complicated by the fact that the relevant high-resolution structures of FucP and LacY are available in dissimilar conformations [Dang et al., 2010; Guan et al., 2007]. In LacY, over 100 residues that have an impact on transport have been identified, but the residue correspondences in FucP, used to argue in favor of the proposed 3-TMS unit rearrangement, are limited to about 15 residues, an important one being an aspartate that influences counterflow [Madej et al., 2013]. "
    [Show abstract] [Hide abstract]
    ABSTRACT: Based on alleged functional residue correspondences between FucP and LacY, a recent study has resulted in a proposed model of 3-TMS unit rearrangements [Madej et al.: Proc Natl Acad Sci USA 2013;110:5870-5874]. We rebut this theory, using 7 different lines of evidence. Our observations suggest that these two transporters are homologous throughout their lengths, having evolved from a common ancestor without repeat unit rearrangements. We exploit the availability of the high-resolution XylE crystal structures in multiple conformations including the inward-facing state to render possible direct comparisons with LacY. Based on a Δdistance map, we confirm the conclusion of Quistgaard et al. [Nat Struct Mol Biol 2013;20:766-768] that the N-terminal 6 TMS halves of these transporters are internally less mobile than the second halves during the conformational transition from the outward occluded state to the inward occluded state and inward occluded state to inward open state. These observations, together with those of Madej et al. [2013], lead to the suggestion that functionally equivalent catalytic residues involved in substrate binding and transport catalysis have evolved in dissimilar positions, but apparently often in similar positions in the putative 3-TMS repeat units, from a single structural scaffold without intragenic rearrangement. © 2014 S. Karger AG, Basel.
    Journal of Molecular Microbiology and Biotechnology 02/2014; 24(2):82-90. DOI:10.1159/000358429 · 1.49 Impact Factor
Show more