Journal of Molecular Biology
Autotransporter β-Domains Have a Specific Function in Protein Secretion beyond Outer-Membrane Targeting
Graphical Abstract
Research Highlights
► AT β-domains are directly involved in passenger translocation. ► They are not just an OM targeting device. ► They are also not a rigid pore that limits passenger translocation.
Introduction
In Gram-negative bacteria, the classical monomeric autotransporter (AT) pathway is a ubiquitous protein secretion system.1 Most characterized ATs are virulence factors. ATs are organized in three domains: (i) an N-terminal signal peptide that targets the protein to the Sec translocon for transport across the inner membrane, (ii) a central passenger domain that is the actual secreted and functional protein, and (iii) a C-terminal β-domain that facilitates the translocation of the passenger domain across the outer membrane (OM). Following transport, the passenger domains of all characterized monomeric ATs are proteolytically separated from their β-domains, after which they either are released into the extracellular environment or remain associated to the membrane via noncovalent interactions, presumably with their β-domain.1
Structural analyses of several secreted passengers of monomeric ATs revealed a common overall topology: they consist of a long stem that consists of an extremely stable right-handed β-helix of triangular rungs to which other functional domains are appended.2, 3, 4, 5 Comprehensive sequence analyses predicted β-helical structures in most of the monomeric ATs, which suggests that the passenger domains of these ATs fold into a similar basic structure.6, 7
The crystal structures of the β-domains of monomeric ATs that have been solved to date also share a common structure: a 12-stranded β-barrel with the α-helical linker inserted into the hydrophilic central pore of the β-barrel.8, 9, 10, 11 The pores have an inner diameter of 10–12 Å and behave very similar in biophysical assays.12 It is generally accepted that the β-domain is required for the translocation of the passenger across the OM. However, whether it functions as the actual translocation pore is under debate for several reasons.1, 13, 14 Firstly, folded elements fused to passenger domains can be transported across the OM.9, 15, 16, 17 These folded domains seem incompatible with the 10-Å diameter of the β-barrel pore, which is just wide enough to accommodate two unfolded polypeptides.8, 18 Secondly, recent cross-linking experiments, using AT passengers that were stalled in the process of OM translocation, revealed contacts with subunits of the β-barrel assembly machinery (Bam) complex,14, 19 which functions in the assembly of OM proteins (OMPs) into the OM (reviewed in Ref. 20).
The potential role of the Bam complex during AT biogenesis has been described by three different models each attributing a different function to the AT β-domain and the Bam complex.21 The insertase model proposes that the Bam complex is only required for the insertion of the AT β-domain into the OM, which would then function as the translocation pore. The translocase model suggests that the Bam complex actively transports the passenger across the OM through a channel inside the Bam complex. In this model, the AT β-domain would primarily serve as a targeting device that delivers the passenger domain to the Bam complex. This is supported by the observations that the major constituent of the Bam complex, BamA, forms a pore in vitro upon interaction with its substrates22 and interacts with the passenger domain of mutants of the AT EspP that were stalled during secretion.19 The third model proposes a concerted function for the Bam complex in β-domain assembly and passenger domain translocation. The Bam machinery would keep the β-domain open in a dilated conformation so that translocation of the passenger can occur.21, 23
In the current study, we investigated whether the β-domain of the AT hemoglobin protease (Hbp) functions in the translocation of its passenger across the OM either as a Bam-targeting device or as a protein-conducting channel. Hbp, which belongs to the serine protease autotransporter of Enterobacteriaceae (SPATE) family, is secreted by a pathogenic strain of Escherichia coli that causes peritonitis.24 Furthermore, it is the only AT for which a crystal structure is available for both the passenger and the β-domain.5, 11 The mature Hbp is released by an intrabarrel autocatalytic mechanism that requires an intimate connection between the α-helical linker and the β-barrel.11, 12 To investigate a putative targeting role, we have substituted the Hbp β-domain for another AT β-domain or for an unrelated integral OMP that is known to interact with the Bam complex. To address the protein-conducting function of the β-domain, we have created Hbp β-barrels that are presumed to form enlarged or narrowed pores. Changes in the pore diameter would affect Hbp secretion if the β-barrel were involved in passenger translocation. The mutants were tested for OM integration, passenger translocation, and secretion. Our results indicate that optimal passenger translocation requires a genuine AT β-domain that carries a 12-stranded β-barrel, suggesting that the β-domain not only functions as a targeting device or as a passive channel for passenger translocation but also is directly involved in its transport.
Section snippets
An AT β-domain is needed for presentation of the passenger at the cell surface
To investigate whether the β-domain functions only as a Bam-targeting device or as a protein-conducting channel as well, we first addressed the targeting function of this domain. β-Barrel proteins are inserted into the OM by the Bam complex.25 Consequently, if the AT β-domain functions only as a Bam-targeting device, we expected that this domain could be replaced by any OMP that folds into a β-barrel. To test this, we substituted the Hbp β-domain by a homologous AT β-domain (EspP) and by an
Discussion
Although the name “autotransporter” suggests an autonomous secretion mechanism, recent work clearly established that ATs interact with the Bam complex during their translocation across the OM.14, 19 The Bam complex inserts integral β-barrel OMPs into the OM37 and was already shown to be indispensable for AT secretion.25, 38 Three models have been proposed for Bam-mediated AT secretion, and these attribute different functions to the AT β-domain. In the insertase model, the AT β-domain functions
Strains, plasmids, and culturing conditions
Strain Top10F′ (Invitrogen) was used for routine cloning and maintenance of plasmid constructs. The strains MC1061 and MC1061degP::S210A29 were used as host for Hbp protein production. The cells were grown at 30 °C in M9 medium, containing 0.1% casamino acids (Difco) and 0.4% glucose. Chloramphenicol (20 μg·ml− 1) was added to the medium when required. The hbp gene and its derivatives (Table 1) were expressed from vector pEH3 as described previously.14
Reagents, enzymes, and sera
Restriction enzymes, T4-DNA ligase,
Acknowledgements
We are grateful to W. Bitter and D. J. Scheffers for helpful discussions and useful comments on the manuscript. We thank V. Koronakis, J. W. de Gier, and I. Rosenkrands for providing antisera. A.S. is supported by NWO Earth and Life Sciences (The Netherlands). Z.S. is supported by an NWO Mosaic grant (The Netherlands). M.S is supported by the Netherlands Leprosy Relief Foundation, the Aeras Global TB Vaccine Foundation, and the Dutch Ministry of Foreign Affairs.
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