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

Escherichia coli is a Gram‐negative bacterium that can use nitrate during anaerobic respiration. The catalytic subunit of the periplasmic nitrate reductase N ap A contains two types of redox cofactor and is exported across the cytoplasmic membrane by the twin‐arginine protein transport pathway. Nap D 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 N ap A . Here we show, using spin labelling and EPR , that the isolated twin‐arginine signal peptide of N ap A is structured in its unbound form and undergoes a small but significant conformational change upon interaction with N ap D . In addition, a complex comprising the full‐length N ap A protein and N ap D could be isolated by engineering an affinity tag onto N ap D only. Analytical ultracentrifugation demonstrated that the two proteins in the N ap DA complex were present in a 1 : 1 molar ratio, and small angle X ‐ray scattering analysis of the complex indicated that N ap A was at least partially folded when bound by its N ap D partner. A N ap DA complex could not be isolated in the absence of the N ap A Tat signal peptide. Taken together, this work indicates that the N ap D chaperone binds primarily at the N ap A signal peptide in this system and points towards a role for N ap D in the insertion of the molybdenum cofactor. Structured digital abstractNapD 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 )