Autotransporter passenger domain secretion requires a hydrophobic cavity at the extracellular entrance of the β-domain pore

Abstract

Whooping cough (pertussis) is a highly contagious acute respiratory illness of humans caused by the Gram-negative bacterial pathogen Bordetella pertussis. The AT (autotransporter) BrkA (Bordetella serum-resistance killing protein A) is an important B. pertussis virulence factor that confers serum resistance and mediates adherence. In the present study, we have solved the crystal structure of the BrkA β-domain at 3 Å (1 Å=0.1 nm) resolution. Special features are a hairpin-like structure formed by the external loop L4, which is observed fortuitously sitting inside the pore of the crystallographic adjacent β-domain, and a previously undiscovered hydrophobic cavity formed by patches on loop L4 and β-strands S5 and S6. This adopts a ubiquitous structure characteristic of all AT β-domains. Mutagenesis studies have demonstrated that the hairpin-like structure and hydrophobic cavity are crucial for BrkA passenger domain (virulence effector) translocation. This structure helps in understanding the molecular mechanism of AT assembly and secretion and provides a potential target for anti-pertussis drug design.

A mortise–tenon joint in the transmembrane domain modulates autotransporter assembly into bacterial outer membranes

“…1d). Pet D1-902 and the Pet D1-902 G 1076 A variant protein were purified in urea and refolded in vitro by rapid dilution of the denaturant in detergent micelles 33 . Pet belongs to the SPATE (serine protease autotransporters of the Enterobacteriaceae) subfamily of autotransporters, which are characterized by an autocatalytic cleavage of the passenger domain, a reaction that depends on precise positioning of the a-linker segment within the lumen of the correctly folded barrel domain 30,31,34 .…”

“…Protein expression and refolding followed previously described methodology 33 . Briefly, E. coli BL21 (DE3) cells transformed with pETPet D1-902 and pETPet D1-902 G 1076 A were harvested after induction with 0.5 mM isopropylthio-b-galactoside, from which inclusion bodies were isolated for protein purification in 8 M urea.…”

“…Folding kinetics of the Pet D1-902 and Pet D1-902 G 1076 A barrel domains were measured after folding was initiated by rapid dilution of the unfolded proteins into detergent micelles at 37°C as described above 33 . Small aliquots of the reaction mixture were removed and quenched with SDS loading buffer (final concentration of SDS: 1% w/v) at the time points indicated, and immediately heated at 100°C.…”

The antigen 43 structure reveals a molecular Velcro-like mechanism of autotransporter-mediated bacterial clumping

“…Despite their abundance and the increasing amount of data linking AT proteins to bacterial virulence, very little structural information is available at a molecular level for these proteins. In fact, AT proteins are significantly underrepresented in the Protein Data Bank (PDB), with only 1 fulllength AT structure, 7 AT passenger domains, 5 AT β-domains, and 12 small domains of trimeric autotransporters deposited among the ∼87,500 structures currently available in the PDB (9,18,(25)(26)(27)(28)(29)(30)(31)(32)(33)(34)(35)(36). None of these entries belong to the large family of AIDA-I-type AT proteins from Gamma-Proteobacteria (3,16).…”

Mechanistic link between β barrel assembly and the initiation of autotransporter secretion

“…β domains are generally ∼30 kDa in size, and although they also display considerable sequence diversity, they can all be identified as members of the pfam03797 (smart00869) family of protein domains. Several divergent β domains have been crystallized and have been shown to form nearly superimposable 12-stranded β barrels that are traversed by an α-helical segment (7)(8)(9)(10). The α-helical segment generally extends into the extracellular space and links the passenger domain to the β domain.…”