Abstract
Autotransporters are bacterial virulence factors that share a common mechanism by which they are transported to the cell surface. They consist of an N-terminal passenger domain and a C-terminal β-barrel, which has been implicated in translocation of the passenger across the outer membrane (OM). The mechanism of passenger translocation and folding is still unclear but involves a conserved region at the C terminus of the passenger domain, the so-called autochaperone domain. This domain functions in the stepwise translocation process and in the folding of the passenger domain after translocation. In the autotransporter hemoglobin protease (Hbp), the autochaperone domain consists of the last rung of the β-helix and a capping domain. To examine the role of this region, we have mutated several conserved aromatic residues that are oriented toward the core of the β-helix. We found that non-conservative mutations affected secretion with Trp(1015) in the cap region as the most critical residue. Substitution at this position yielded a DegP-sensitive intermediate that is located at the periplasmic side of the OM. Further analysis revealed that Trp(1015) is most likely required for initiation of processive folding of the β-helix at the cell surface, which drives sequential translocation of the Hbp passenger across the OM.
Highlights
Contains the virulence function and a conserved C-terminal domain that forms a -barrel pore in the outer membrane (OM), as implied by the original model for secretion [4, 5]
The C-terminal region of the AT passenger domain just upstream of the -domain has been implicated in the folding and secretion across the OM, but the precise function of this so-called AC domain has remained unclear [22]
We have identified in the SPATE hemoglobin protease (Hbp) a conserved Trp at position 1015 that is critical for translocation of the passenger across the OM
Summary
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]. It has been suggested that BamA may act directly as the protein-conducting channel for the AT passenger or indirectly by coordinating the assembly of the -domain and translocation of the passenger in a concerted fashion [30, 31] In the latter model, the -domain may still function as the actual channel for passenger translocation, assuming that it is accommodated in or near the Bam complex in an extended translocation-competent conformation that transiently forms during transport of the passenger. The data are consistent with a role for the C-terminal region of the -helix in the initiation of processive folding of the -helix at the cell surface to drive sequential translocation of the Hbp passenger across the OM
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