© 2015 by The American Society for Biochemistry and Molecular Biology, Inc. 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 sugarbinding site and might participate in sugar binding through apolar interactions.
|Journal||Journal of Biological Chemistry|
|Publication status||Published - 26 Jun 2015|
Fuerst, O., Lin, Y., Granell, M., Leblanc, G., Padrós, E., Lórenz-Fonfría, V. A., & Cladera, J. (2015). The melibiose transporter of Escherichia coli: Critical contribution of Lys-377 to the structural organization of the interacting substrate binding sites. Journal of Biological Chemistry, 290(26), 16261-16271. https://doi.org/10.1074/jbc.M115.642678