In bacteria the formation of disulphide bonds is facilitated by a family of enzymes known as the disulphide bond forming (Dsb) proteins, which, despite low sequence homology, belong to the thioredoxin (TRX) superfamily. Among these enzymes is the disulphide bond-forming protein A (DsbA); a periplasmic thiol oxidase responsible for catalysing the oxidative folding of numerous cell envelope and secreted proteins [1]. This work encompasses the structural and functional analysis of FtDsbA1, a DsbA homologue from the highly pathogenic Gram-negative bacterium Francisella tularensis.
Our analysis shows that FtDsbA1 shares a conserved TRX-like fold, characterised by a core βαβ-α-ββα scaffold, bridged by an alpha helical bundle showcased by all DsbA-like proteins. However, FtDsbA1 displays a highly unique variation on this structure, containing an extended and flexible N-terminus and secondary structural elements inserted within the core of the TRX fold itself, which together twist the overall DsbA-like architecture. While modifications to the N and the C terminus of the TRX core fold are common, FtDsbA1 represents one of the only examples of a TRX-like protein with multiple additional helices embedded within the ββα motif of the TRX domain itself. Additionally, FtDsbA1 exhibits variations to the well conserved DsbA active site with an unusual dipeptide in the catalytic CXXC redox centre (CGKC), and a trans configuration for the singularly conserved cis-proline loop, known for governing DsbA-substrate interactions [2], which likely accounts for FtDsbA1’s narrow substrate specificity.
Overall, the structure of FtDsbA1 not only represents a structurally unique DsbA enzyme, but also underscores the remarkable malleability of the TRX catalytic core- a ubiquitous and ancestral protein fold.