title: Characterization of a periplasmic nitrate reductase in complex with its biosynthetic chaperone.
abstract: 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.
authors: Division of Molecular Microbiology, College of Life Sciences, University of Dundee, UK.
authors: Jennifer M
authors: JM
authors: Dow
authors: Sabine
authors: S
authors: Grahl
authors: Richard
authors: R
authors: Ward
authors: Rachael
authors: R
authors: Evans
authors: Olwyn
authors: O
authors: Byron
authors: David G
authors: DG
authors: Norman
authors: Tracy
authors: T
authors: Palmer
authors: Frank
authors: F
authors: Sargent
mesh_terms: D002352:Carrier Proteins / Q000378:metabolism*; D004578:Electron Spin Resonance Spectroscopy; D004926:Escherichia coli / Q000378:metabolism*; D029968:Escherichia coli Proteins / Q000378:metabolism*; D007501:Iron / Q000378:metabolism; D018832:Molecular Chaperones / Q000378:metabolism*; D050901:Nitrate Reductase / Q000378:metabolism*; D009566:Nitrates / Q000378:metabolism; D010084:Oxidation-Reduction; D019897:Periplasm / Q000378:metabolism*; D011485:Protein Binding; D011994:Recombinant Proteins / Q000378:metabolism*; D053838:Scattering, Small Angle; D014461:Ultracentrifugation
chemical_list: D002352:Carrier Proteins; D029968:Escherichia coli Proteins; D018832:Molecular Chaperones; C524414:NapD protein, E coli; D009566:Nitrates; D011994:Recombinant Proteins; D007501:Iron; D050901:Nitrate Reductase; C494404:napA protein, E coli
keywords: Tat pathway; chaperone; periplasmic nitrate reductase; protein-protein interaction; twin-arginine signal peptide