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

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.