Characterisation of a pre-export enzyme-chaperone complex on the twin-arginine transport pathway

Jennifer M Dow, Frank Gabel, Frank Sargent (Lead / Corresponding author), Tracy Palmer (Lead / Corresponding author)

    Research output: Contribution to journalArticle

    14 Citations (Scopus)

    Abstract

    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.
    Original languageEnglish
    Pages (from-to)57-66
    Number of pages10
    JournalBiochemical Journal
    Volume452
    Issue number1
    Early online date1 Mar 2013
    DOIs
    Publication statusPublished - 15 May 2013

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