Structure and Mechanism of a Nitrate Transporter

Abstract

The nitrate/nitrite transporters NarK and NarU play an important role in nitrogen homeostasis in bacteria and belong to the nitrate/nitrite porter family (NNP) of the major facilitator superfamily (MFS) fold. The structure and functional mechanism of NarK and NarU remain unknown. Here, we report the crystal structure of NarU at a resolution of 3.1 Å and systematic biochemical characterization. The two molecules of NarU in an asymmetric unit exhibit two distinct conformational states: occluded and partially inward-open. The substrate molecule nitrate appears to be coordinated by four highly conserved, charged, or polar amino acids. Structural and biochemical analyses allowed the identification of key amino acids that are involved in substrate gating and transport. The observed conformational differences of NarU, together with unique sequence features of the NNP family transporters, suggest a transport mechanism that might deviate from the canonical rocker-switch model.

Transferable Denitrification Capability of Thermus thermophilus

“…NarK1-like transporters, such as NarK from T. thermophilus NAR1, are proposed to be nitrate/proton symporters, whereas NarK2 transporters, like NarT transporters, are proposed to be nitrate/nitrite antiporters (61,62). Recently, the structures of the two NNP transporters of E. coli that belong to the NarK2 subfamily, NarU and NarK, have been solved (63,64). Based on the structure of NarU, a model was proposed in which nitrate transport is catalyzed by subtle conformational changes in a group of internal membrane helices facilitating transition from an "outward-open" state that allows the entrance of the anion from the periplasm into its binding site in the center of the protein and into the "inward-open" state that allows the exit of the anion to the cytoplasm.…”

Legacy Nutrient Dynamics at the Watershed Scale: Principles, Modeling, and Implications

“…Such accumulation would be more considerable in smallholder cropping systems. Smallholder farmers in China as well as in other regions usually apply livestock manure only considering the N contained in the manure but without considering the P contained in the manure (Han et al, 2012;Yan et al, 2013), resulting in excessive P application when coupled with synthetic fertilizer P additions and P accumulation in cropland soils. As estimated previously, cropland soil legacy P pools could be 504-953 kg P ha À1 in the China over the past three decades (Hou et al, 2013;Jiang and Yuan, 2015;Li et al, 2015).…”

Structural basis for dynamic mechanism of proton-coupled symport by the peptide transporter POT

“…The strict coupling of the structural transition and the binding of the substrate and H + to the transporter enable the substrate uptake to be driven by the energy stored in the electrochemical gradient across the membrane. The previously determined structures of MFS members in the inward-open [LacY,, (6)(7)(8) and GlpT (9)], inward-occluded [PiPT (10)], occluded [EmrD (11) and NarU (12)], outward-occluded [XylE (13)], and outward-open [FucP (14)] conformations provided structural evidence for this alternating-access mechanism. However, the means by which the structural transition and the substrate binding are coupled, thus forming the basis of the symport mechanism of MFS, still remain elusive (15).…”