Sugar Binding in Lactose Permease: Anomeric State of a Disaccharide Influences Binding Structure

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Journal of Molecular Biology (Impact Factor: 4.33). 05/2007; 367(5):1523-34. DOI: 10.1016/j.jmb.2007.02.001
Source: PubMed


Lactose permease in Escherichia coli (LacY) transports both anomeric states of disaccharides but has greater affinity for alpha-sugars. Molecular dynamics (MD) simulations are used to probe the protein-sugar interactions, binding structures, and global protein motions in response to sugar binding by investigating LacY (the experimental mutant and wild-type) embedded in a fully hydrated lipid bilayer. A total of 12 MD simulations of 20-25 ns each with beta(alpha)-d-galactopyranosyl-(1,1)-beta-d-galactopyranoside (betabeta-(Galp)(2)) and alphabeta-(Galp)(2) result in binding conformational families that depend on the anomeric state of the sugar. Both sugars strongly interact with Glu126 and alphabeta-(Galp)(2) has a greater affinity to this residue. Binding conformations are also seen that involve protein residues not observed in the crystal structure, as well as those involved in the proton translocation (Phe118, Asn119, Asn240, His322, Glu325, and Tyr350). Common to nearly all protein-sugar structures, water acts as a hydrogen bond bridge between the disaccharide and protein. The average binding energy is more attractive for alphabeta-(Galp)(2) than betabeta-(Galp)(2), i.e. -10.7(+/-0.7) and -3.1(+/-1.0) kcal/mol, respectively. Of the 12 helices in LacY, helix-IV is the least stable with betabeta-(Galp)(2) binding resulting in larger distortion than alphabeta-(Galp)(2).

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Available from: Jeffery Klauda
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    • "Additional mutations in LacY that result in the enhancement of transport for other distinct sugars have been found and are well-documented (Brooker, Fiebig et al. 1985; Brooker and Wilson 1985; Brooker and Wilson 1985; Collins, Permuth et al. 1989; Franco, Eelkema et al. 1989; Olsen and Brooker 1989; Brooker 1990; King and Wilson 1990; Brooker 1991; Eelkema, O'Donnell et al. 1991; Gram and Brooker 1992; Goswitz and Brooker 1993; Olsen, Greene et al. 1993; Varela and Wilson 1996; Varela, Brooker et al. 1997; Varela, Wilson et al. 2000; Shinnick and Varela 2002). Computational and biophysical analyses have been useful in the elucidation of sugar binding properties and of the conformational changes that occur during lactose transport across the membrane (Park and Lee 2005; Kasho, Smirnova et al. 2006; Vadyvaloo, Smirnova et al. 2006; Yin, Jensen et al. 2006; Jensen, Yin et al. 2007; Klauda and Brooks 2007). In addition, studies of maltoporin (LamB) have been tremendously helpful in the elucidation the molecular basis of maltose transport across the membrane via outer membrane porin channels (Boos and Shuman 1998). "
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    ABSTRACT: In order to identify amino acid residues in the Escherichia coli raffinose-H+ permease (RafB) that play a role in sugar selection and transport, we first incubated E. coli HS4006 containing plasmid pRU600 (expresses inducible raffinose permease and α-galactosidase) on maltose MacConkey indicator plates overnight. Initially, all colonies were white, indicating no fermentation of maltose. Upon further incubation, 100 mutants appeared red. pRU600 DNA was prepared from 55 mutants. Five mutants transferred the phenotype for fermentation of maltose (red). Plasmid DNA from five maltose-positive phenotype transformants was prepared and sequenced, revealing three distinct types of mutations. Two mutants exhibited Val-35→Ala (MT1); one mutant had Ile-391→Ser (MT2); and two mutants had Ser-138→Asp, Ser-139→Leu and Gly-389→Ala (MT3). Transport studies of [3H]-maltose showed that cells harboring MT1, MT2 and MT3 had greater uptake (P ≤ 0.05) than cells harboring wild-type RafB. However, [14C]-raffinose uptake was reduced in all mutant cells (P ≤ 0.05) with MT1, MT2 and MT3 mutants compared to cells harboring wild-type RafB. Kinetic analysis showed enhanced apparent K m values for maltose and reduced V max/ K m ratios for raffinose compared to wild-type values. The apparent K i value of maltose for RafB indicates a competitive relationship between maltose and raffinose. Maltose “uphill” accumulation was greater for mutants (P ≤ 0.05) than for cells with wild-type RafB. Thus, we implicate residues in RafB that are responsible for raffinose transport and suggest that the substituted residues in RafB dictate structures that enhance transport of maltose.
    Full-text · Article · Dec 2007 · Journal of Membrane Biology
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    ABSTRACT: Molecular dynamics simulations of lactose permease (LacY) in a phospholipid bilayer reveal the conformational dynamics of the protein. In inhibitor-bound simulations (i.e., those closest to the X-ray structure) the protein was stable, showing little conformational change over a 50 ns timescale. Movement of the bound inhibitor, TDG, to an alternative binding mode was observed, so that it interacted predominantly with the N-terminal domain and with residue E269 from the C-terminal domain. In multiple ligand-free simulations, a degree of domain closure occurred. This switched LacY to a state with a central cavity closed at both the intracellular and periplasmic ends. This may resemble a possible intermediate in the transport mechanism. Domain closure occurs by a combination of rigid-body movements of domains and of intradomain motions of helices, especially TM4, TM5, TM10, and TM11. A degree of intrahelix flexibility appears to be important in the conformational change.
    Preview · Article · Aug 2007 · Structure
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    ABSTRACT: 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.
    Preview · Article · Oct 2007 · Proceedings of the National Academy of Sciences
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