Vectorial transport and folding of an autotransporter virulence protein during outer membrane secretion

Abstract

We introduced pairs of cysteine residues into the passenger sequence of pertactin, an AT virulence protein from Bordetella pertussis, and show that OM secretion of the passenger domain stalls due to the formation of a disulphide bond. We further show that the C-terminus of the pertactin passenger domain b-helix crosses the OM first, followed by the N-terminal portions of the virulence protein. In vivo proteolytic digestion shows that the C-terminus is exposed to the extracellular milieu during stalling, and forms stable structure. These AT secretion and folding features can potentially facilitate efficient secretion.

A Conserved Aromatic Residue in the Autochaperone Domain of the Autotransporter Hbp Is Critical for Initiation of Outer Membrane Translocation

“…This would be expected for a sequential role of the aromatic residues in the core during the C-to N-terminal folding of the ␤-helix region at the cell surface after a hairpin in the ␤-domain is formed. In accordance with this supposition, for an intermediate of pertactin that was stalled during translocation halfway across the passenger domain, it was shown that the C terminus had crossed the OM (27).…”

“…However, most of the available evidence suggests that translocation of the passenger is a vectorial process in which the ␤-domain functions as a protein-conducting channel (2, 3). Recent in vitro unfolding/refolding studies revealed that the pertactin ␤-helix refolds to native protein via an intermediate state with a folded C terminus and unfolded N terminus (20,27). In Hbp, the Trp 1015 residue is located in the cap region that folds to cover the C-terminal end of the ␤-helical stem of the passenger (Fig.…”

“…Deletion and mutagenesis experiments have shown that this region is crucial for efficient folding of several passenger domains (23)(24)(25)(26). Recent evidence indicates that translocation of the passenger across the OM occurs in an C-to-N direction, possibly following the formation of a hairpin structure by the C terminus of the passenger in the ␤-domain channel (27,28). In this scenario, the AC domain might function as a template for stacking of the ␤-helix to initiate folding (22).…”

Size and Conformation Limits to Secretion of Disulfide-bonded Loops in Autotransporter Proteins

“…Studies investigating this relationship between the folding and secretion of AT passenger domains have largely focused on disulfide bond formation during transit through the periplasm, between exogenous Cys residues introduced into passenger domains (20,28), or between Cys residues within heterologous passenger domains (29 -36). Although the translocation of sizable folded domains comprising disulfidebonded segments of heterologous passenger domains was reported (30 -32), a greater number of studies have demonstrated that periplasmic formation of either long disulfidebonded loops or tightly folded/rigid structures is incompatible with OM translocation/secretion of AT passenger domains (20,28,29,(33)(34)(35)(36). These dichotomous sets of studies have given rise to two mechanistic models of AT biogenesis, one proposing that passenger domain translocation occurs through a folded AT ␤-barrel pore, and the other proposing BamA as an accessory factor mediating passenger translocation before the ␤-domain adopts a final folded conformation.…”

Subdomain 2 of the Autotransporter Pet Is the Ligand Site for Recognizing the Pet Receptor on the Epithelial Cell Surface

“…Usually, the ␤-helix forms a backbone to which additional, functional subdomains are attached. This ␤-helical structure is capped by a C-terminal region, which emerges into the medium first (15). This region forms a structure unique to the AT family and is known as the junction or "autochaperone" (AC) due to its inferred role in folding the entire passenger.…”