Crystal structure of a nitrate/nitrite exchanger

Abstract

Mineral nitrogen in nature is often found in the form of nitrate (NO3(-)). Numerous microorganisms evolved to assimilate nitrate and use it as a major source of mineral nitrogen uptake. Nitrate, which is central in nitrogen metabolism, is first reduced to nitrite (NO2(-)) through a two-electron reduction reaction. The accumulation of cellular nitrite can be harmful because nitrite can be reduced to the cytotoxic nitric oxide. Instead, nitrite is rapidly removed from the cell by channels and transporters, or reduced to ammonium or dinitrogen through the action of assimilatory enzymes. Despite decades of effort no structure is currently available for any nitrate transport protein and the mechanism by which nitrate is transported remains largely unknown. Here we report the structure of a bacterial nitrate/nitrite transport protein, NarK, from Escherichia coli, with and without substrate. The structures reveal a positively charged substrate-translocation pathway lacking protonatable residues, suggesting that NarK functions as a nitrate/nitrite exchanger and that protons are unlikely to be co-transported. Conserved arginine residues comprise the substrate-binding pocket, which is formed by association of helices from the two halves of NarK. Key residues that are important for substrate recognition and transport are identified and related to extensive mutagenesis and functional studies. We propose that NarK exchanges nitrate for nitrite by a rocker switch mechanism facilitated by inter-domain hydrogen bond networks.

Fig. 1. The crystal structure of NarK

a. Part of TM2 of NarK is shown in stereo view with a sigma-weighted 2F_o-F_c map at 2.6Å resolution, contoured at 1.0 σ. b. Left, NarK structure viewed from the plane of membrane with the putative location of the lipid bilayer as indicated. NarK is colored in rainbow with the N-terminus in blue. Right, NarK viewed from the periplasmic side. The identity of the 12 transmembrane helices is indicated. c. The N-terminal domain (TM1-6) of NarK (blue) is psudo-symmetric to the C-terminal domain (TM7-12) (yellow) and can be superimposed with an r.m.s.d of 2.9Å. d. Cut-away surface representation of the inward-facing NarK shows the central cavity exposed to the cytosol.

Fig. 2. The substrate-binding site in NarK

a. Two highly conserved NS motifs in TM5 and TM11 (blue helices) at the center of NarK form the nitrate/nitrite transport pathway. b. The substrate-binding pocket is defined by two evolutionarily conserved and functionally important arginine residues R89 and R305. The binding site is capped above and below by F267 and F147, respectively. c. R89 and R305 are stabilized by inter-domain H-bonds as depicted. The two halves of NarK are indicated as blue (N-terminal domain) and yellow (C-terminal domain). d. Nitrite bound structure of NarK. The density for nitrite is observed at the substrate-binding site after soaking the NarK crystals with sodium nitrite. R305 undergoes a conformational change upon substrate binding. This conformational change affects the inter-domain network of H-bonds between Y263, G171 and R305. The displayed map is a sigmaA-weighted F_o-F_c at 3σ.

Fig. 3. Protons are likely excluded from the substrate translocation pathway of NarK

a. Location of histidine, aspartate and glutamate residues in the anion transporter GlpT. Acidic residues line the substrate translocation pathway. b. Electrostatic surface representation for each domain of GlpT showing a relatively even distribution of positive and negative charges in the substrate translocation pathway. c. Location of histidine, aspartate and glutamate residues in the fucose transporter FucP. Acidic residues line the substrate translocation pathway. d. Electrostatic surface representation for each domain of FucP. e. Location of histidine, aspartate and glutamate residues in NarK. No acidic residues are found in the substrate translocation pathway of NarK. f. Electrostatic surface representation for NarK showing a dominantly positively charged substrate translocation pathway. It represents a formidable barrier for the translocation of H⁺ but could attract negatively charged molecules like nitrate and nitrite.

Fig. 4. Proposed mechanism of nitrate/nitrite exchange

Six conformations of NarK are depicted as a. outward facing, b. outward facing with nitrate bound, c. occluded with nitrate bound, d. inward facing with nitrate release, e. inward facing with nitrite bound, f. occluded with nitrite bound. Once NarK completes the cycle and returns to the outward facing conformation nitrite is released to the periplasm. The proposed mechanism is based on the rocker switch. For NarK the rocker switch is facilitated by breaking and reforming inter-domain H-bonds involving R89 and R305 as described in Fig 2.