title: The Melibiose Transporter of Escherichia coli: CRITICAL CONTRIBUTION OF LYS-377 TO THE STRUCTURAL ORGANIZATION OF THE INTERACTING SUBSTRATE BINDING SITES.
abstract: We examine the role of Lys-377, the only charged residue in helix XI, on the functional mechanism of the Na(+)-sugar melibiose symporter from Escherichia coli. Intrinsic fluorescence, FRET, and Fourier transform infrared difference spectroscopy reveal that replacement of Lys-377 with either Cys, Val, Arg, or Asp disables both Na(+) and melibiose binding. On the other hand, molecular dynamics simulations extending up to 200-330 ns reveal that Lys-377 (helix XI) interacts with the anionic side chains of two of the three putative ligands for cation binding (Asp-55 and Asp-59 in helix II). When Asp-59 is protonated during the simulations, Lys-377 preferentially interacts with Asp-55. Interestingly, when a Na(+) ion is positioned in the Asp-55-Asp-59 environment, Asp-124 in helix IV (a residue essential for melibiose binding) reorients and approximates the Asp-55-Asp-59 pair, and all three acidic side chains act as Na(+) ligands. Under these conditions, the side chain of Lys-377 interacts with the carboxylic moiety of these three Asp residues. These data highlight the crucial role of the Lys-377 residue in the spatial organization of the Na(+) binding site. Finally, the analysis of the second-site revertants of K377C reveals that mutation of Ile-22 (in helix I) preserves Na(+) binding, whereas that of melibiose is largely abolished according to spectroscopic measurements. This amino acid is located in the border of the sugar-binding site and might participate in sugar binding through apolar interactions.
authors.affiliation: From the Unitat de Biofísica, Departament de Bioquímica i de Biologia Molecular, Facultat de Medicina, and Centre d'Estudis en Biofísica, Universitat Autònoma de Barcelona, 08193 Bellaterra, Barcelona, Spain.
authors.forename: Oliver
authors.initials: O
authors.lastname: Fuerst
authors.affiliation: From the Unitat de Biofísica, Departament de Bioquímica i de Biologia Molecular, Facultat de Medicina, and Centre d'Estudis en Biofísica, Universitat Autònoma de Barcelona, 08193 Bellaterra, Barcelona, Spain.
authors.forename: Yibin
authors.initials: Y
authors.lastname: Lin
authors.affiliation: From the Unitat de Biofísica, Departament de Bioquímica i de Biologia Molecular, Facultat de Medicina, and Centre d'Estudis en Biofísica, Universitat Autònoma de Barcelona, 08193 Bellaterra, Barcelona, Spain.
authors.forename: Meritxell
authors.initials: M
authors.lastname: Granell
authors.affiliation: the Direction des Sciences du Vivant, Direction des progammes et valorization, CEA Fontenay-aux-Roses, 92265 Fontenay-aux-Roses Cedex, France, and.
authors.forename: Gérard
authors.initials: G
authors.lastname: Leblanc
authors.affiliation: From the Unitat de Biofísica, Departament de Bioquímica i de Biologia Molecular, Facultat de Medicina, and Centre d'Estudis en Biofísica, Universitat Autònoma de Barcelona, 08193 Bellaterra, Barcelona, Spain.
authors.forename: Esteve
authors.initials: E
authors.lastname: Padrós
authors.affiliation: From the Unitat de Biofísica, Departament de Bioquímica i de Biologia Molecular, Facultat de Medicina, and Centre d'Estudis en Biofísica, Universitat Autònoma de Barcelona, 08193 Bellaterra, Barcelona, Spain, Experimental Molecular Biophysics, Department of Physics, Freie Universität Berlin, 14195 Berlin, Germany.
authors.forename: Víctor A
authors.initials: VA
authors.lastname: Lórenz-Fonfría
authors.affiliation: From the Unitat de Biofísica, Departament de Bioquímica i de Biologia Molecular, Facultat de Medicina, and Centre d'Estudis en Biofísica, Universitat Autònoma de Barcelona, 08193 Bellaterra, Barcelona, Spain, josep.cladera@uab.cat.
authors.forename: Josep
authors.initials: J
authors.lastname: Cladera
mesh_terms: D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D001665:Binding Sites; D004926:Escherichia coli / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D007532:Isoleucine / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D007700:Kinetics; D008239:Lysine / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D008553:Melibiose / Q000737:chemistry / Q000378:metabolism; D056004:Molecular Dynamics Simulation; D012964:Sodium / Q000737:chemistry / Q000378:metabolism; D027981:Symporters / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*
chemical_list: D027981:Symporters; D007532:Isoleucine; C021548:melibiose permease; D008553:Melibiose; D012964:Sodium; D008239:Lysine
keywords: FRET; IR spectroscopy; membrane biophysics; molecular dynamics; secondary transporter; sodium ligands; sugar transport