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

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.

Integrative omics identification, evolutionary and structural analysis of low affinity nitrate transporters in diatoms, diNPFs.

Diatoms are one of the major and most diverse groups of phytoplankton, with chimeric genomes harbouring a combination of genes of bacterial, animal and plant origin. They have developed sophisticated mechanisms to face environmental variations. In marine environments, nutrients concentration shows significant temporal and spatial variability, influencing phytoplankton growth. Among nutrients, nitrogen, present at micromolar levels, is often a limiting resource. Here, we report a comprehensive characterization of the Nitrate Transporter 1/Peptide Transporter Family (NPF) in diatoms, diNPFs. NPFs are well characterized in many organisms where they recognize a broad range of substrates, ranging from short-chained di- and tri-peptides in bacteria, fungi and mammals to a wide variety of molecules including nitrate in higher plants. Scarce information is available for diNPFs. We integrated-omics, phylogenetic, structural and expression analyses, to infer information on their role in diatoms. diNPF genes diverged to produce two distinct clades with strong sequence and structural homology with either bacterial or plant NPFs, with different predicted sub-cellular localization, suggesting that the divergence resulted in functional diversification. Moreover, transcription analysis of diNPF genes under different laboratory and environmental growth conditions suggests that diNPF diversification led to genetic adaptations that might contribute to diatoms ability to flourish in diverse environmental conditions.