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

• Published in: The FEBS journal Vol. 281; no. 1; pp. 246 - 260
• Main Authors: Dow, Jennifer M., Grahl, Sabine, Ward, Richard, Evans, Rachael, Byron, Olwyn, Norman, David G., Palmer, Tracy, Sargent, Frank
• Format: Journal Article
• Language: English
• Published: England Blackwell Publishing Ltd 01.01.2014; Published by Blackwell Pub. on behalf of the Federation of European Biochemical Societies
• Subjects: Biosynthesis; Biotechnology; Carrier Proteins - metabolism; chaperone; E coli; Electron Spin Resonance Spectroscopy; Escherichia coli - metabolism; Escherichia coli Proteins - metabolism; Iron - metabolism; Molecular Chaperones - metabolism; Nitrate Reductase - metabolism; Nitrates; Nitrates - metabolism; Original; Oxidation-Reduction; Periplasm - metabolism; periplasmic nitrate reductase; Protein Binding; Protein folding; protein–protein interaction; Recombinant Proteins - metabolism; Scattering, Small Angle; Tat pathway; twin‐arginine signal peptide; Ultracentrifugation; protein-protein interaction; Tat pathway; periplasmic nitrate reductase; chaperone; twin-arginine signal peptide
• ISSN: 1742-464X; 1742-4658
• DOI: 10.1111/febs.12592

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. Structured digital NapD and NapA bind by x ray scattering (View interaction) NapA and NapD physically interact by molecular sieving (View interaction) NapA and NapD bind by electron paramagnetic resonance (View interaction) The bacterial periplasmic nitrate reductase, NapA, is bound tightly during its biosynthesis by the NapD chaperone. Here, pulsed EPR, analytical ultracentrifugation, and small angle X‐ray scattering were used to characterise the complex. This work indicates that the NapD chaperone binds primarily at the NapA signal peptide, and the proteins are present in the NapDA complex in a 1 : 1 molar ratio.