Publications

• How do antiporters exchange substrates across the cell membrane? An atomic-level description of the complete exchange cycle in NarK.

  Feng J., Selvam B., Shukla D.

  Major facilitator superfamily (MFS) proteins operate via three different mechanisms: uniport, symport, and antiport. Despite extensive investigations, the molecular understanding of antiporters is less advanced than that of other transporters due to the complex coupling between two substrates and ... >> More

  Major facilitator superfamily (MFS) proteins operate via three different mechanisms: uniport, symport, and antiport. Despite extensive investigations, the molecular understanding of antiporters is less advanced than that of other transporters due to the complex coupling between two substrates and the lack of distinct structures. We employ extensive all-atom molecular dynamics simulations to dissect the complete substrate exchange cycle of the bacterial NO₃^-/NO₂^- antiporter, NarK. We show that paired basic residues in the binding site prevent the closure of unbound protein and ensure the exchange of two substrates. Conformational transition occurs only in the presence of substrate, which weakens the electrostatic repulsion and stabilizes the transporter. Furthermore, we propose a state-dependent substrate exchange model, in which the relative spacing between the paired basic residues determines whether NO₃^- and NO₂^- bind simultaneously or sequentially. Overall, this work presents a general working model for the antiport mechanism within the MFS. << Less

  Structure 29:922-933.e3(2021) [PubMed] [EuropePMC]

• A single channel for nitrate uptake, nitrite export and nitrite uptake by Escherichia coli NarU and a role for NirC in nitrite export and uptake.

  Jia W., Tovell N., Clegg S., Trimmer M., Cole J.

  Two related polytopic membrane proteins of the major facilitator family, NarK and NarU, catalyse nitrate uptake, nitrite export and nitrite uptake across the Escherichia coli cytoplasmic membrane by an unknown mechanism. A 12-helix model of NarU was constructed based upon six alkaline phosphatase ... >> More

  Two related polytopic membrane proteins of the major facilitator family, NarK and NarU, catalyse nitrate uptake, nitrite export and nitrite uptake across the Escherichia coli cytoplasmic membrane by an unknown mechanism. A 12-helix model of NarU was constructed based upon six alkaline phosphatase and beta-galactosidase fusions to NarK and the predicted hydropathy for the NarK family. Fifteen residues conserved in the NarK-NarU protein family were substituted by site-directed mutagenesis, including four residues that are essential for nitrate uptake by Aspergillus nidulans: arginines Arg(87) and Arg(303) in helices 2 and 8, and two glycines in a nitrate signature motif. Despite the wide range of substitutions studied, in no case did mutation result in loss of one biochemical function without simultaneous loss of all other functions. A NarU+ NirC+ strain grew more rapidly and accumulated nitrite more rapidly than the isogenic NarU+ NirC(-) strain. Only the NirC+ strain consumed nitrite rapidly during the later stages of growth. Under conditions in which the rate of nitrite reduction was limited by the rate of nitrite uptake, NirC+ strains reduced nitrite up to 10 times more rapidly than isogenic NarU+ strains, indicating that both nitrite efflux and nitrite uptake are largely dependent on NirC. Isotope tracer experiments with [15N]nitrate and [14N]nitrite revealed that [15N]nitrite accumulated in the extracellular medium even when there was a net rate of nitrite uptake and reduction. We propose that NarU functions as a single channel for nitrate uptake and nitrite expulsion, either as a nitrate-nitrite antiporter, or more likely as a nitrate/H+ or nitrite/H+ channel. << Less

  Biochem. J. 417:297-304(2009) [PubMed] [EuropePMC]

  This publication is cited by 1 other entry.