Quaternary structure of a SPATE autotransporter protein

Hritonenko, Victoria; Kostakioti, Maria; Stathopoulos, Christos

doi:10.1080/09687860600821316

Cited by 13 publications

(12 citation statements)

References 35 publications

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“…This could only be the case if the conserved linker motif folded in an ␣-helix while still in the periplasm and participated in interactions that drove secretion of the passenger through the pore prior to its own transport. However, this (11). These findings are in agreement with other studies indicating that the EspP, AIDA, and NalP ATs are monomeric (17,22,27).…”

Section: Discussionsupporting

confidence: 83%

Role of the α-Helical Linker of the C-Terminal Translocator in the Biogenesis of the Serine Protease Subfamily of Autotransporters

Kostakioti

Stathopoulos

2006

Infect Immun

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Autotransporters are secreted virulence factors that comprise three domains: an N-terminal signal peptide, an internal passenger domain, and a C-terminal ␤-domain. The mechanism of passenger translocation across the outer membrane remains undefined, with four models having been proposed: the "hairpin," the "threading," the "multimeric," and the "Omp85 (YaeT)" models. In an attempt to understand autotransporter biogenesis, we screened the sequences of the serine protease subfamily of autotransporters (SPATEs) for conserved features indicative of a common secretion mechanism. Our analyses revealed a strictly conserved 14-amino-acid motif within the predicted ␣-helical linker region, upstream of the ␤-domain of SPATEs. We investigated the function of this motif through a mutagenesis approach using Tsh as a model. Our studies demonstrate that mutations throughout the conserved motif do not block insertion of the ␤-domain into the outer membrane. However, nonconservative mutations of four hydrophobic (V1099, L1102, G1107, and L1109) and three polar (N1100, K1104, and R1105) residues of the motif severely decrease or even abolish Tsh biogenesis. Further studies showed that these mutations interfere with passenger transport across the outer membrane. Bioinformatical analyses suggest that the critical polar and hydrophobic amino acids localize on opposite sides of the helix that runs through the ␤-barrel pore. Our data indicate that the conserved motif is important for passenger secretion across the outer membrane and that mutations in certain residues severely affect the secretion process. We discuss how these results fit with the four proposed models for autotransporter secretion and potential applications in antimicrobial and vaccine development.

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Section: Discussionsupporting

confidence: 83%

Role of the α-Helical Linker of the C-Terminal Translocator in the Biogenesis of the Serine Protease Subfamily of Autotransporters

Kostakioti

Stathopoulos

2006

Infect Immun

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“…Based on the observation that the deletion of the ␤ domain abolishes passenger domain secretion, it was originally proposed that the ␤ domain functions as the transporter for the covalently linked passenger domain (29). Because translocation proceeds in a C-to N-terminal direction (13,16), and most (if not all) ␤ domains are monomeric (11,20,22,31), translocation would almost certainly be initiated by the insertion of a C-terminal segment of the passenger domain into the ␤ domain pore in a hairpin conformation. Subsequently, more N-terminal segments of the passenger domain would slide progressively past a static strand.…”

mentioning

confidence: 99%

Residues in a Conserved α-Helical Segment Are Required for Cleavage but Not Secretion of an Escherichia coli Serine Protease Autotransporter Passenger Domain

Dautin

Bernstein

2011

J Bacteriol

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Autotransporters are a superfamily of virulence factors produced by Gram-negative bacteria that are comprised of an N-terminal extracellular domain (passenger domain) and a C-terminal ␤ barrel domain (␤ domain) that resides in the outer membrane (OM). The ␤ domain promotes the translocation of the passenger domain across the OM by an unknown mechanism. Available evidence indicates that an ␣-helical segment that spans the passenger domain-␤ domain junction is embedded inside the ␤ domain at an early stage of assembly. Following its secretion, the passenger domain of the serine protease autotransporters of the Enterobacteriaceae (SPATEs) and the pertactin family of Bordetella pertussis autotransporters is released from the ␤ domain through an intrabarrel autoproteolytic cleavage of the ␣-helical segment. Although the mutation of conserved residues that surround the cleavage site has been reported to impair both the translocation and cleavage of the passenger domain of a SPATE called Tsh, we show here that the mutation of the same residues in another SPATE (EspP) affects only passenger domain cleavage. Our results strongly suggest that the conserved residues are required to position the ␣-helical segment for the cleavage reaction and are not required to promote passenger domain secretion.

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“…It seems clear that multimerization is not a conserved feature of the AT C-terminal domains. Monomeric structures have been reported for the crystal structures of NalP, EspP, and EstA (2,41,59), and monomers and dimers have been detected using biochemical methods for EspP (53), AIDA-I (37), and Tsh (18).…”

Section: Discussionmentioning

confidence: 99%

Comparative Analysis of the Biochemical and Functional Properties of C-Terminal Domains of Autotransporters

2010

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Autotransporters (ATs) are the largest group of proteins secreted by Gram-negative bacteria and include many virulence factors from human pathogens. ATs are synthesized as large precursors with a C-terminal domain that is inserted in the outer membrane (OM) and is essential for the translocation of an N-terminal passenger domain to the extracellular milieu. Several mechanisms have been proposed for AT secretion. Self-translocation models suggest transport across a hydrophilic channel formed by an internal pore of the ␤-barrel or by the oligomerization of C-terminal domains. Alternatively, an assisted-translocation model suggests that transport employs a conserved machinery of the bacterial OM such as the Bam complex. In this work we have investigated AT secretion by carrying out a comparative study to analyze the conserved biochemical and functional features of different C-terminal domains selected from ATs of gammaproteobacteria, betaproteobacteria, alphaproteobacteria, and epsilonproteobacteria. Our results indicate that C-terminal domains having an N-terminal ␣-helix and a ␤-barrel constitute functional transport units for the translocation of peptides and immunoglobulin domains with disulfide bonds. In vivo and in vitro analyses show that multimerization is not a conserved feature in AT C-terminal domains. Furthermore, we demonstrate that the deletion of the conserved ␣-helix severely impairs ␤-barrel folding and OM insertion and thereby blocks passenger domain secretion. These observations suggest that the AT ␤-barrel without its ␣-helix cannot form a stable hydrophilic channel in the OM for protein translocation. The implications of our data for an understanding of AT secretion are discussed.The classical autotransporter (AT) family, also known as the type Va protein secretion system, represents the largest group of proteins secreted by Gram-negative bacteria and includes many virulence factors from important human pathogens (10, 17). Bacteria produce AT proteins as large polypeptide precursors, with their virulence activity (e.g., cytotoxins, adhesins, and proteases, etc.) present in a passenger domain flanked by an N-terminal signal peptide (sp) for Sec-dependent translocation across the bacterial inner membrane (IM) and a Cterminal domain of ϳ30 to 40 kDa for insertion into the bacterial outer membrane (OM) (see Fig. 2A). A self-translocation model was originally proposed to explain the secretion mechanism of AT proteins across the OM, based mostly on data obtained with the IgA protease (IgAP) from Neisseria gonorrhoeae (43). In this model the C-terminal domain of ATs was supposed to fold in the OM as a ␤-barrel protein with an internal hydrophilic pore that could be used for the translocation of the passenger domain. The finding that the B subunit of cholera toxin (CtxB) should not have disulfide bonds for its secretion when fused as a heterologous passenger to the Cterminal domain of IgAP (30, 31) indirectly suggests passenger translocation in an unfolded conformation through a narrow channel expected...

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Quaternary structure of a SPATE autotransporter protein

Cited by 13 publications

References 35 publications

Role of the α-Helical Linker of the C-Terminal Translocator in the Biogenesis of the Serine Protease Subfamily of Autotransporters

Role of the α-Helical Linker of the C-Terminal Translocator in the Biogenesis of the Serine Protease Subfamily of Autotransporters

Residues in a Conserved α-Helical Segment Are Required for Cleavage but Not Secretion of an Escherichia coli Serine Protease Autotransporter Passenger Domain

Comparative Analysis of the Biochemical and Functional Properties of C-Terminal Domains of Autotransporters

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