Energetics and mechanism of anion permeation across formate-nitrite transporters

Abstract

Formate-nitrite transporters (FNTs) facilitate the translocation of monovalent polyatomic anions, such as formate and nitrite, across biological membranes. FNTs are widely distributed among pathogenic bacteria and eukaryotic parasites, but they lack human homologues, making them attractive drug targets. The mechanisms and energetics involved in anion permeation across the FNTs have remained largely unclear. Both, channel and transporter mode of function have been proposed, with strong indication of proton coupling to the permeation process. We combine molecular dynamics simulations, quantum mechanical calculations, and pK _a calculations, to compute the energetics of the complete permeation cycle of an FNT. We find that anions as such, are not able to traverse the FNT pore. Instead, anion binding into the pore is energetically coupled to protonation of a centrally located histidine. In turn, the histidine can protonate the permeating anion, thereby enabling its release. Such mechanism can accommodate the functional diversity among the FNTs, as it may facilitate both, export and import of substrates, with or without proton co-transport. The mechanism excludes proton leakage via the Grotthuss mechanism, and it rationalises the selectivity for weak acids.

Figure 1

Crystal structure of a NirC monomer (PDB ID: 4FC4). The side chains of the constriction-forming residues and of the central histidine are shown as sticks and labeled in the zoomed image. Pore representation done with HOLE, colour coded by pore radius: red < 1 Å < green < 2.5 Å < blue.

Figure 2

PMFs for permeation of different substrates (see legend) across NirC, HSC, VcFocA, and EcFocA (from left to right), calculated using umbrella sampling. Top row: permeation of anions across a pore with a neutral central histidine (HIS0), middle row: permeation of anions and water across a pore with a positively-charged central histidine (HIS+), bottom row: permeation of neutral substrates and water across a HIS0 pore. The tan and brown bars indicate the cytoplasmic and periplasmic constriction, respectively. Anions experience a high barrier for permeation across the HIS0 pore, and strong binding into the HIS+ pore.

Figure 3

Mutual stabilisation of a formate and hydronium ion entering the pore. The curves denote the difference between (i) the sum-of-distances PMF for simultaneous internalisation of the ions ΔG _sum(ξ), and (ii) the sum of the single-ion PMFs of formate and a classical hydronium model, while considering different compartments of origin of the ions: (A) both ions enter from the periplasmic space, (B) the hydronium and formate ion enter from the periplasmic and cytoplasmic space, respectively, (C) the hydronium and formate ion enter from the cytoplasmic and periplasmic space, respectively.

Figure 4

Proton transfer between formate (a,b) or nitrite (c,d) and the central histidine in the NirC central chamber (a,c) or a capped histidine residue in bulk water solution (b,d). The distances of the proton to the donor atom (N_δ atom of the histidine), and to the acceptor atom (an oxygen atom of formate or nitrite) are shown vs. time (raw trace and running average). In the protein environment, frequent proton jumps between the central histidine and the bound anion are observed on a picosecond time scale. (e) Simulation snapshot from the central chamber, illustrating the distances plotted in the graphs. Atoms of the QM region are shown as spheres.

Figure 5

Permeation of formate across NirC. Shown are the PMFs for permeation of formate across a HIS+ pore (black) and for permeation of formic acid across a HIS0 pore (red), corrected for the respective free energies of protonation of the central histidine in the protein (black arrow on the right), and of formate in bulk assuming pH = 7 and taking the formic acid pK _a of 3.75 (red arrow on the right). The arrow on the top denotes the main physiologically-relevant direction of permeation across NirC. Once the anion is bound to the central histidine (z ≈ 0 at the black curve), it can be quickly protonated by it (jumping from the black to the red PMF at z ≈ 0), after which the substrate is enabled to leave the pore more easily, with lowest barrier towards the cytoplasm.

Figure 6

Permeation mechanism including (a) simultaneous internalisation of anion (A⁻) and proton (H⁺) into the pore, (b) anion protonation by the central histidine into its neutral counterpart (HA), and (c) release of the neutral substrate from the pore. This mechanism allows for anion import or export in an electrogenic channel-like manner (bottom branch) or as proton symport (top branch).