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

Lookup NU author(s): Professor Tracy Palmer, Professor Frank Sargent

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This work is licensed under a Creative Commons Attribution 4.0 International License (CC BY 4.0).


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. © 2013 The Authors.


Publication metadata

Author(s): Dow JM, Grahl S, Ward R, Evans R, Byron O, Norman DG, Palmer T, Sargent F

Publication type: Article

Publication status: Published

Journal: FEBS Journal

Year: 2014

Volume: 281

Issue: 1

Pages: 246-260

Print publication date: 01/01/2014

Online publication date: 04/11/2013

Acceptance date: 28/10/2013

Date deposited: 15/02/2019

ISSN (print): 1742-464X

ISSN (electronic): 1742-4658

Publisher: Wiley-Blackwell Publishing Ltd.

URL: https://doi.org/10.1111/febs.12592

DOI: 10.1111/febs.12592


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