Journal
JOURNAL OF BIOLOGICAL CHEMISTRY
Volume 290, Issue 26, Pages 16261-16271Publisher
AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC
DOI: 10.1074/jbc.M115.642678
Keywords
FRET; IR spectroscopy; membrane biophysics; molecular dynamics; sugar transport; secondary transporter; sodium ligands
Categories
Funding
- Ministerio de Ciencia e Innovacion
- Fondo Europeo de Desarrollo Regional Grant [BFU2012-40137-C02--01]
- the Commissariat a l'Energie Atomique (CEA-Saclay)
- Ministerio de Educacion, Cultura y Deporte Fellowship [3729]
- Universitat Autonoma de Barcelona FPI fellowship
- Universitat Autonoma de Barcelona Postdoctoral Fellowship [40607]
- Marie Curie Reintegration Grant [PIRG03--6A-2008--231063]
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Background: Lys-377 mutants of the E. coli melibiose permease are defective in the cotransport of substrates. Results: Lys-377 mutants do not bind substrates, whereas Ile-22 mutants bind Na+ but not sugars. Both observations are explained by molecular dynamics simulations. Conclusion: Lys-377 is crucial for the organization of the binding site. Ile-22 may participate in sugar binding. Significance: Uncovering the architecture of the substrate binding site is essential for understanding the transport mechanism. 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.
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