The twin-arginine translocation (Tat) system is a protein targeting pathway utilised by prokaryotes and chloroplasts. Tat substrates are produced with distinctive N-terminal signal peptides and are translocated as fully folded proteins. In Escherichia coli Tat-dependent proteins often contain redox cofactors that must be loaded before translocation. Trimethylamine N-oxide reductase (TorA) is a model bacterial Tat substrate and is a molybdenum cofactor-dependent enzyme. Co-ordination of cofactor loading and translocation of TorA is directed by the TorD protein, which is a cytoplasmic chaperone known to physically interact with the TorA signal peptide. In this work, a pre-export TorAD complex has been characterised using biochemical and biophysical techniques, including SAXS. A stable, cofactor-free TorAD complex was isolated, which revealed a 1:1 binding stoichiometry. Surprisingly, a TorAD complex with similar architecture can be isolated in the complete absence of the 39-residue TorA signal peptide. This work demonstrates that two high-affinity binding sites for TorD are present on TorA, and that a single TorD protein binds both of those simultaneously. Further characterisation suggested that the C-terminal 'Domain IV' of TorA remained solvent-exposed in the cofactor-free pre-export TorAD complex. It is possible that correct folding of Domain IV upon cofactor loading is the trigger for TorD release and subsequent export of TorA.
Dow, J. M., Gabel, F., Sargent, F., & Palmer, T. (2013). Characterisation of a pre-export enzyme-chaperone complex on the twin-arginine transport pathway. Biochemical Journal, 452(1), 57-66. https://doi.org/10.1042/BJ20121832