title: Characterization of a pre-export enzyme-chaperone complex on the twin-arginine transport pathway.
abstract: The Tat (twin-arginine translocation) system is a protein targeting pathway utilized 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 interact physically with the TorA signal peptide. In the present study, a pre-export TorAD complex has been characterized using biochemical and biophysical techniques, including SAXS (small-angle X-ray scattering). 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. The present study 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 characterization 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.
authors: Division of Molecular Microbiology, College of Life Sciences, University of Dundee, Dundee DD1 5EH, UK.
authors: Jennifer M
authors: JM
authors: Dow
authors: Frank
authors: F
authors: Gabel
authors: Frank
authors: F
authors: Sargent
authors: Tracy
authors: T
authors: Palmer
mesh_terms: D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics / Q000378:metabolism; D029968:Escherichia coli Proteins / Q000737:chemistry* / Q000235:genetics / Q000502:physiology; D026901:Membrane Transport Proteins / Q000737:chemistry* / Q000235:genetics; D018832:Molecular Chaperones / Q000737:chemistry* / Q000235:genetics / Q000502:physiology; D010089:Oxidoreductases, N-Demethylating / Q000737:chemistry* / Q000235:genetics / Q000502:physiology; D011485:Protein Binding / Q000235:genetics; D021382:Protein Sorting Signals / Q000235:genetics; D021381:Protein Transport / Q000235:genetics; D014178:Translocation, Genetic
chemical_list: D029968:Escherichia coli Proteins; D026901:Membrane Transport Proteins; D018832:Molecular Chaperones; D021382:Protein Sorting Signals; C088083:TorD protein, E coli; C408585:twin-arginine translocase complex, E coli; D010089:Oxidoreductases, N-Demethylating; C023186:trimethylamine dehydrogenase