Allosteric regulation of nitrate transporter NRT via the signaling protein PII

Proc Natl Acad Sci U S A. 2024 Mar 12;121(11):e2318320121. doi: 10.1073/pnas.2318320121. Epub 2024 Mar 8.

Abstract

Coordinated carbon and nitrogen metabolism is crucial for bacteria living in the fluctuating environments. Intracellular carbon and nitrogen homeostasis is maintained by a sophisticated network, in which the widespread signaling protein PII acts as a major regulatory hub. In cyanobacteria, PII was proposed to regulate the nitrate uptake by an ABC (ATP-binding cassette)-type nitrate transporter NrtABCD, in which the nucleotide-binding domain of NrtC is fused with a C-terminal regulatory domain (CRD). Here, we solved three cryoelectron microscopy structures of NrtBCD, bound to nitrate, ATP, and PII, respectively. Structural and biochemical analyses enable us to identify the key residues that form a hydrophobic and a hydrophilic cavity along the substrate translocation channel. The core structure of PII, but not the canonical T-loop, binds to NrtC and stabilizes the CRD, making it visible in the complex structure, narrows the substrate translocation channel in NrtB, and ultimately locks NrtBCD at an inhibited inward-facing conformation. Based on these results and previous reports, we propose a putative transport cycle driven by NrtABCD, which is allosterically inhibited by PII in response to the cellular level of 2-oxoglutarate. Our findings provide a distinct regulatory mechanism of ABC transporter via asymmetrically binding to a signaling protein.

Keywords: ABC transporter; carbon and nitrogen homeostasis; nitrate/nitrite transport; signaling protein PII; single-particle cryogenic electron microscopy.

MeSH terms

  • Adenosine Triphosphate / metabolism
  • Allosteric Regulation
  • Bacterial Proteins / metabolism
  • Carbon / metabolism
  • Cryoelectron Microscopy
  • Cyanobacteria* / metabolism
  • Nitrate Transporters*
  • Nitrates / metabolism
  • Nitrogen / metabolism
  • PII Nitrogen Regulatory Proteins / genetics
  • PII Nitrogen Regulatory Proteins / metabolism

Substances

  • Nitrate Transporters
  • Nitrates
  • Bacterial Proteins
  • Adenosine Triphosphate
  • Nitrogen
  • Carbon
  • PII Nitrogen Regulatory Proteins