Characterization of a periplasmic nitrate reductase in complex with its biosynthetic chaperone

Jennifer M. Dow, Sabine Grahl, Richard Ward, Rachael Evans, Olwyn Byron, David G Norman, Tracy Palmer (Lead / Corresponding author), Frank Sargent (Lead / Corresponding author)

Research output: Contribution to journalArticlepeer-review

8 Citations (Scopus)
152 Downloads (Pure)


Escherichia coli is a Gram-negative bacterium that can use nitrate during anaerobic respiration. The catalytic subunit of the periplasmic nitrate reductase NapA contains two types of redox cofactor and is exported across the cytoplasmic membrane by the twin-arginine protein transport pathway. NapD is a small cytoplasmic protein that is essential for the activity of the periplasmic nitrate reductase and binds tightly to the twin-arginine signal peptide of NapA. Here we show, using spin labelling and EPR, that the isolated twin-arginine signal peptide of NapA is structured in its unbound form and undergoes a small but significant conformational change upon interaction with NapD. In addition, a complex comprising the full-length NapA protein and NapD could be isolated by engineering an affinity tag onto NapD only. Analytical ultracentrifugation demonstrated that the two proteins in the NapDA complex were present in a 1 : 1 molar ratio, and small angle X-ray scattering analysis of the complex indicated that NapA was at least partially folded when bound by its NapD partner. A NapDA complex could not be isolated in the absence of the NapA Tat signal peptide. Taken together, this work indicates that the NapD chaperone binds primarily at the NapA signal peptide in this system and points towards a role for NapD in the insertion of the molybdenum cofactor.
Original languageEnglish
Pages (from-to)246-260
Number of pages15
JournalFEBS Journal
Issue number1
Early online date4 Nov 2013
Publication statusPublished - 9 Dec 2013


  • chaperone
  • periplasmic nitrate reductase
  • protein-protein interaction
  • Tat pathway
  • twin-arginine signal peptide

ASJC Scopus subject areas

  • Biochemistry
  • Cell Biology
  • Molecular Biology


Dive into the research topics of 'Characterization of a periplasmic nitrate reductase in complex with its biosynthetic chaperone'. Together they form a unique fingerprint.

Cite this