Autotransporter structure reveals intra-barrel cleavage followed by conformational changes

Abstract

Autotransporters are virulence factors produced by Gram-negative bacteria. They consist of two domains, an N-terminal 'passenger' domain and a C-terminal beta-domain. beta-domains form beta-barrel structures in the outer membrane while passenger domains are translocated into the extracellular space. In some autotransporters, the two domains are separated by proteolytic cleavage. Using X-ray crystallography, we solved the 2.7-A structure of the post-cleavage state of the beta-domain of EspP, an autotransporter produced by Escherichia coli strain O157:H7. The structure consists of a 12-stranded beta-barrel with the passenger domain-beta-domain cleavage junction located inside the barrel pore, approximately midway between the extracellular and periplasmic surfaces of the outer membrane. The structure reveals an unprecedented intra-barrel cleavage mechanism and suggests that two conformational changes occur in the beta-domain after cleavage, one conferring increased stability on the beta-domain and another restricting access to the barrel pore.

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

“…Cell extracts were then heated in SDS/PAGE sample buffer and the cleaved EspP β domain was detected by Western blot using an antibody generated against a C-terminal peptide. Consistent with previous results (8), the wild-type β domain was resistant to SDS-denaturation when heated up to 70°C (Fig. 2, first blot).…”

“…1 A and B). Based on the crystal structure of the EspP β domain (8), the side chain of any amino acid larger than glycine at position 1066 (including the methyl group of an alanine residue) is predicted to project into the lumen of the β barrel and clash sterically with the side chain of a second highly conserved residue, W1042 ( Fig. 1 B and C, and Fig.…”

“…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.…”

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

“…In EspP 29,30 [pdb 2QOM] and Hbp 31 [pdb 3AEH] the mortise motif maps to b-strand number 3 of the barrel domain, while a tenon motif maps to b-strand number 4 (Fig. 1c).…”

“…Pet was the first identified autotransporter with enterotoxic activity and is a critical virulence factor of enteroaggregative Escherichia coli 32 . The homologous protein from shiga toxin-producing Escherichia coli, called EspP 29,30 , has been crystallized thereby providing a means to model the topology of Pet (Supplementary Fig. 1a) and three-dimensional structure of Pet ( Supplementary Fig.…”