The X-ray structure of the type II secretion system complex formed by the N-terminal domain of EpsE and the cytoplasmic domain of EpsL of Vibrio cholerae

Gram-negative bacteria use type II secretion systems for the transport of virulence factors and hydrolytic enzymes through the outer membrane. These sophisticated multi-protein complexes reach from the pore in the outer membrane via the pseudopilins in the periplasm and a multi-protein inner-membrane sub-complex, to an ATPase in the cytoplasm. The human pathogen Vibrio cholerae uses such a secretion machinery, called the Eps-system, for the export of its major virulence factor cholera toxin into the intestinal tract of the human host. Here, we describe the 2.4 A structure of the hetero-tetrameric complex of the N-terminal domain of the ATPase EpsE and the cytoplasmic domain of the inner membrane protein EpsL, which constitute the major cytoplasmic components of the Eps-system. A stable fragment of EpsE in complex with the cytoplasmic domain of EpsL was identified via limited proteolysis and facilitated the crystallization of the complex. This first structure of a complex between two different proteins of the type II secretion system reveals that the N-terminal domain of EpsE and the cytoplasmic domain of EpsL form a hetero-tetramer, in which EpsL is the central dimer and EpsE binds on the periphery. The dimer of EpsL in this complex is very similar to the dimer seen in the crystal structure of the native cytoplasmic domain of EpsL, suggesting a possible physiological relevance despite a relatively small 675 A2 buried solvent accessible surface. The N-terminal domain of EpsE, which forms a compact domain with an alpha+beta-fold, places its helix alpha2 in a mostly hydrophobic cleft between domains II and III of EpsL burying 1700 A2 solvent accessible surface. This extensive interface involves several residues whose hydrophobic or charged nature is well conserved and is therefore likely to be of general importance in type II secretion systems.     

Synergistic stimulation of EpsE ATP hydrolysis by EpsL and acidic phospholipids

EpsE is a cytoplasmic component of the type II secretion system in Vibrio cholerae. Through ATP hydrolysis and an interaction with the cytoplasmic membrane protein EpsL, EpsE supports secretion of cholera toxin across the outer membrane. In this study, we have determined the effect of the cytoplasmic domain of EpsL (cyto-EpsL) and purified phospholipids on the ATPase activity of EpsE. Acidic phospholipids, specifically cardiolipin, bound the copurified EpsE/cyto-EpsL complex and stimulated its ATPase activity 30-130-fold, whereas the activity of EpsE alone was unaffected. Removal of the last 11 residues (residues 243-253) from cyto-EpsL prevented cardiolipin binding as well as stimulation of the ATPase activity of EpsE. Further mutagenesis of the C-terminal region of the EpsL cytoplasmic domain adjacent to the predicted transmembrane helix suggested that this region participates in fine tuning the interaction of EpsE with the cytoplasmic membrane and influences the oligomerization state of EpsE thereby stimulating its ATPase activity and promoting extracellular secretion in V. cholerae.     

In vivo cross-linking of EpsG to EpsL suggests a role for EpsL as an ATPase-pseudopilin coupling protein in the Type II secretion system of Vibrio cholerae

The type II secretion system is a multi-protein complex that spans the cell envelope of Gram-negative bacteria and promotes the secretion of proteins, including several virulence factors. This system is homologous to the type IV pilus biogenesis machinery and contains five proteins, EpsG-K, termed the pseudopilins that are structurally homologous to the type IV pilins. The major pseudopilin EpsG has been proposed to form a pilus-like structure in an energy-dependent process that requires the ATPase, EpsE. A key remaining question is how the membrane-bound EpsG interacts with the cytoplasmic ATPase, and if this is a direct or indirect interaction. Previous studies have established an interaction between the bitopic inner membrane protein EpsL and EpsE; therefore, in this study we used in vivo cross-linking to test the hypothesis that EpsG interacts with EpsL. Our findings suggest that EpsL may function as a scaffold to link EpsG and EpsE and thereby transduce the energy generated by ATP hydrolysis to support secretion. The recent discovery of structural homology between EpsL and a protein in the type IV pilus system implies that this interaction may be conserved and represent an important functional interaction for both the type II secretion and type IV pilus systems.     

Interaction between the autokinase EpsE and EpsL in the cytoplasmic membrane is required for extracellular secretion in Vibrio cholerae.

Vibrio cholerae secretes a number of proteins important for virulence, including cholera toxin. This process requires the products of the eps genes which have homologues in genera such as Aeromonas, Klebsiella and Pseudomonas and are thought to form a membrane-associated multiprotein complex. Here we show that the putative nucleotide-binding protein EpsE is associated with and stabilized by the cytoplasmic membrane via interaction with EpsL. Analysis of fusion proteins between EpsE and the homologous ExeE from Aeromonas hydrophila demonstrates that the N-terminus of EpsE contains the EpsL binding domain and determines species specificity. An intact Walker A box, commonly found in ATP-binding proteins, is required for activity of EpsE in vivo and for autophosphorylation of purified EpsE in vitro. These results indicate that both the kinase activity of EpsE as well as its ability to interact with the putative cytoplasmic membrane protein EpsL are required for translocation of toxin across the outer membrane in Vibrio cholerae.     

Two regions of EpsL involved in species-specific protein-protein interactions with EpsE and EpsM of the general secretion pathway in Vibrio cholerae

Extracellular secretion of proteins via the type II or general secretion pathway in gram-negative bacteria requires the assistance of at least 12 gene products that are thought to form a complex apparatus through which secreted proteins are translocated. Although this apparatus is specifically required only for the outer membrane translocation step during transport across the bacterial cell envelope, it is believed to span both membranes. The EpsE, EpsL, and EpsM proteins of the type II apparatus in Vibrio cholerae are thought to form a trimolecular complex that is required to either control the opening and closing of the secretion pore or to transduce energy to the site of outer membrane translocation. EpsL is likely to play an important role in this relay by interacting with both the cytoplasmic EpsE protein and the cytoplasmic membrane protein EpsM, which is predominantly exposed on the periplasmic side of the membrane. We have now extended this model and mapped the separate regions within EpsL that contain the EpsE and EpsM binding domains. By taking advantage of the species specificity of the type II pathway, we have used chimeric proteins composed of EpsL and its homologue, ExeL, from Aeromonas hydrophila together with either EpsE or its Aeromonas homologue, ExeE, to complement the secretion defect in both epsL and exeL mutant strains. These studies have mapped the species-specific EpsE binding site to the N-terminal cytoplasmic region between residues 57 and 216 of EpsL. In addition, the species-specific EpsM binding site was mapped to the C-terminal half of EpsL by coimmunoprecipitation of EpsM with different EpsL-ExeL chimeras. This site is present in the region between amino acids 216 and 296, which contains the predicted membrane-spanning segment of EpsL.     

Direct Interaction of the EpsL and EpsM Proteins of the General Secretion Apparatus in Vibrio cholerae

The general secretion pathway of gram-negative bacteria is responsible for extracellular secretion of a number of different proteins, including proteases and toxins. This pathway supports secretion of proteins across the cell envelope in two distinct steps, in which the second step, involving translocation through the outer membrane, is assisted by at least 13 different gene products. Two of these components, the cytoplasmic membrane proteins EpsL and EpsM of Vibrio cholerae, have been purified and characterized. Based on gel filtration analysis, both purified EpsM(His)6 and wild-type EpsL present in an Escherichia coli Triton X-100 extract are dimeric proteins. EpsL and EpsM were also found to interact directly and form a Triton X-100 stable complex that could be precipitated with either anti-EpsL or anti-EpsM antibodies. In addition, when the L and M proteins were coexpressed in E. coli, they formed a stable complex and protected each other from proteolytic degradation, indicating that these two proteins interact in vivo and that no other Eps protein is required for their association. Since EpsL is predicted to contain a large cytoplasmic domain, while EpsM is predominantly exposed on the periplasmic side, we speculate that these components might be part of a structure that is involved in bridging the inner and outer membranes. Furthermore, since EpsL has previously been shown to interact with the autophosphorylating cytoplasmic membrane protein EpsE, we hypothesize that this trimolecular complex might be involved in regulating the opening and closing of the secretion pore and/or transducing energy to the site of outer membrane translocation.     

Molecular analysis of the Vibrio cholerae type II secretion ATPase EpsE

The type II secretion system is a macromolecular assembly that facilitates the extracellular translocation of folded proteins in gram-negative bacteria. EpsE, a member of this secretion system in Vibrio cholerae, contains a nucleotide-binding motif composed of Walker A and B boxes that are thought to participate in binding and hydrolysis of ATP and displays structural homology to other transport ATPases. Here we demonstrate that purified EpsE is an Mg2+-dependent ATPase and define optimal conditions for the hydrolysis reaction. EpsE displays concentration-dependent activity, which may suggest that the active form is oligomeric. Size exclusion chromatography showed that the majority of purified EpsE is monomeric; however, detailed analyses of specific activities obtained following gel filtration revealed the presence of a small population of active oligomers. We further report that EpsE binds zinc through a tetracysteine motif near its carboxyl terminus, yet metal displacement assays suggest that zinc is not required for catalysis. Previous studies describing interactions between EpsE and other components of the type II secretion pathway together with these data further support the hypothesis that EpsE functions to couple energy to the type II apparatus, thus enabling secretion.     

Oligomerization of EpsE coordinates residues from multiple subunits to facilitate ATPase activity

EpsE is an ATPase that powers transport of cholera toxin and hydrolytic enzymes through the Type II secretion (T2S) apparatus in the gram-negative bacterium, Vibrio cholerae. On the basis of structures of homologous Type II/IV secretion ATPases and our biochemical data, we believe that EpsE is active as an oligomer, likely a hexamer, and the binding, hydrolysis, and release of nucleotide cause EpsE to undergo dynamic structural changes, thus converting chemical energy to mechanical work, ultimately resulting in extracellular secretion. The conformational changes that occur as a consequence of nucleotide binding would realign conserved arginines (Arg(210), Arg(225), Arg(320), Arg(324), Arg(336), and Arg(369)) from adjoining domains and subunits to complete the active site around the bound nucleotide. Our data suggest that these arginines are essential for ATP hydrolysis, although their roles in shaping the active site of EpsE are varied. Specifically, we have shown that replacements of these arginine residues abrogate the T2S process due to a reduction of ATPase activity yet do not have any measurable effect on nucleotide binding or oligomerization of EpsE. We have further demonstrated that point mutations in the EpsE intersubunit interface also reduce ATPase activity without disrupting oligomerization, strengthening the idea that residues from multiple subunits must precisely interact in order for EpsE to be sufficiently active to support T2S. Our findings suggest that the action of EpsE is similar to that of other Type II/IV secretion ATPase family members, and thus these results may be widely applicable to the family as a whole.     

Structure of an essential type IV pilus biogenesis protein provides insights into pilus and type II secretion systems

Type IV pili (T4Ps) are long cell surface filaments, essential for microcolony formation, tissue adherence, motility, transformation, and virulence by human pathogens. The enteropathogenic Escherichia coli bundle-forming pilus is a prototypic T4P assembled and powered by BfpD, a conserved GspE secretion superfamily ATPase held by inner-membrane proteins BfpC and BfpE, a GspF-family membrane protein. Although the T4P assembly machinery shares similarity with type II secretion (T2S) systems, the structural biochemistry of the T4P machine has been obscure. Here, we report the crystal structure of the two-domain BfpC cytoplasmic region (N-BfpC), responsible for binding to ATPase BfpD and membrane protein BfpE. The N-BfpC structure reveals a prominent central cleft between two α/β-domains. Despite negligible sequence similarity, N-BfpC resembles PilM, a cytoplasmic T4P biogenesis protein. Yet surprisingly, N-BfpC has far greater structural similarity to T2S component EpsL, with which it also shares virtually no sequence identity. The C-terminus of the cytoplasmic domain, which leads to the transmembrane segment not present in the crystal structure, exits N-BfpC at a positively charged surface that most likely interacts with the inner membrane, positioning its central cleft for interactions with other Bfp components. Point mutations in surface-exposed N-BfpC residues predicted to be critical for interactions among BfpC, BfpE, and BfpD disrupt pilus biogenesis without precluding interactions with BfpE and BfpD and without affecting BfpD ATPase activity. These results illuminate the relationships between T4P biogenesis and T2S systems, imply that subtle changes in component residue interactions can have profound effects on function and pathogenesis, and suggest that T4P systems may be disrupted by inhibitors that do not preclude component assembly.     

The 1.59 Å resolution structure of the minor pseudopilin EpsH of Vibrio cholerae reveals a long flexible loop

The type II secretion complex exports folded proteins from the periplasm to the extracellular milieu. It is used by the pathogenic bacterium Vibrio cholerae to export several proteins, including its major virulence factor, cholera toxin. The pseudopilus is an essential component of the type II secretion system and likely acts as a piston to push the folded proteins across the outer membrane through the secretin pore. The pseudopilus is composed of the major pseudopilin, EpsG, and four minor pseudopilins, EpsH, EpsI, EpsJ and EpsK. We determined the x-ray crystal structure of the head domain of EpsH at 1.59 Å resolution using molecular replacement with the previously reported EpsH structure, 2qv8, as the template. Three additional N-terminal amino acids present in our construct prevent an artifactual conformation of residues 160–166, present in one of the two monomers of the 2qv8 structure. Additional crystal contacts stabilize a long flexible loop comprised of residues 104–135 that is more disordered in the 2qv8 structure but is partially observed in our structure in very different positions for the two EpsH monomers in the asymmetric unit. In one of the conformations the loop is highly extended. Modeling suggests the highly charged loop is capable of contacting EpsG and possibly secreted protein substrates, suggesting a role in specificity of pseudopilus assembly or secretion function.     

Function-Related Positioning of the Type II Secretion ATPase of Xanthomonas campestris pv. campestris

Gram-negative bacteria use the type II secretion (T2S) system to secrete exoproteins for attacking animal or plant cells or to obtain nutrients from the environment. The system is unique in helping folded proteins traverse the outer membrane. The secretion machine comprises multiple proteins spanning the cell envelope and a cytoplasmic ATPase. Activity of the ATPase, when copurified with the cytoplasmic domain of an interactive ATPase partner, is stimulated by an acidic phospholipid, suggesting the membrane-associated ATPase is actively engaged in secretion. How the stimulated ATPase activity is terminated when secretion is complete is unclear. We fused the T2S ATPase of Xanthomonas campestris pv. campestris, the causal agent of black rot in the crucifers, with fluorescent protein and found that the ATPase in secretion-proficient cells was mainly diffused in cytoplasm. Focal spots at the cell periphery were detectable only in a few cells. The discrete foci were augmented in abundance and intensity when the secretion channel was depleted and the exoprotein overproduced. The foci abundance was inversely related to secretion efficiency of the secretion channel. Restored function of the secretion channel paralleled reduced ATPase foci abundance. The ATPase foci colocalized with the secretion channel. The ATPase may be transiently associated with the T2S machine by alternating between a cytoplasmic and a machine-associated state in a secretion-dependent manner. This provides a logical means for terminating the ATPase activity when secretion is completed. Function-related dynamic assembly may be the essence of the T2S machine.     

Protein secretion in the absence of ATP: the autotransporter,two-partner secretion and chaperone/usher pathways of Gram-negative bacteria (Review)

Bacteria secrete a wide variety of proteins, many of which play important roles in virulence. In Gram-negative bacteria, these proteins must cross the cytoplasmic or inner membrane, periplasm, and outer membrane to reach the cell surface. Gram-negative bacteria have evolved multiple pathways to allow protein secretion across their complex envelope. ATP is not available in the periplasm and many of these secretion pathways encode components that harness energy available at the inner membrane to drive secretion across the outer membrane. In contrast, the autotransporter, two-partner secretion and chaperone/usher pathways are comparatively simple systems that allow secretion across the outer membrane without the need for input of energy from the inner membrane. This review will present overviews of these ‘self-sufficient’ pathways, focusing on recent advances and secretion mechanisms. Similarities among the pathways and with other protein translocation mechanisms will be highlighted.     

Protein secretion in the absence of ATP: the autotransporter, two-partner secretion and chaperone/usher pathways of gram-negative bacteria (review)

Bacteria secrete a wide variety of proteins, many of which play important roles in virulence. In gram-negative bacteria, these proteins must cross the cytoplasmic or inner membrane, periplasm, and outer membrane to reach the cell surface. Gram-negative bacteria have evolved multiple pathways to allow protein secretion across their complex envelope. ATP is not available in the periplasm and many of these secretion pathways encode components that harness energy available at the inner membrane to drive secretion across the outer membrane. In contrast, the autotransporter, two-partner secretion and chaperone/usher pathways are comparatively simple systems that allow secretion across the outer membrane without the need for input of energy from the inner membrane. This review will present overviews of these 'self-sufficient' pathways, focusing on recent advances and secretion mechanisms. Similarities among the pathways and with other protein translocation mechanisms will be highlighted.     

Structural and functional studies of EpsC, a crucial component of the type 2 secretion system from Vibrio cholerae

The type 2 secretion system (T2SS) occurring in Gram-negative bacteria is composed of 12-15 different proteins which form large assemblies spanning two membranes and secreting several virulence factors in folded state across the outer membrane. The T2SS component EpsC of Vibrio cholerae plays an important role in this machinery. While anchored in the inner membrane, by far the largest part of EpsC is periplasmic, containing a so-called homology region (HR) domain and a PDZ domain. Here we report studies on the structure and function of both periplasmic domains of EpsC. The crystal structures of two variants of the PDZ domain of EpsC from V. cholerae were determined at better than 2 A resolution. Compared to the short variant, the longer variant contains an additional N-terminal helix, and reveals a significant difference in the position of helix alphaB with respect to the beta-sheet. Both our structures show that the PDZ domain of EpsC adopts a more open form than in previously reported structures of other PDZ domains. Most interestingly, in the crystals of the short EpsC-PDZ domain the peptide binding groove interacts with an alpha-helix from a neighboring subunit burying approximately 921 A2 solvent accessible surface. This makes it possible that the PDZ domain of this bacterial protein binds proteins in a manner which is altogether different from that seen in any other PDZ domain so far. We also determined that the HR domain of EpsC is primarily responsible for the interaction with the secretin EpsD, while the PDZ is not, or much less, so. This new finding, together with studies of others, leads to the suggestion that the PDZ domain of EpsC may interact with exoproteins to be secreted while the HR domain plays a key role in linking the inner-membrane sub-complex of the T2SS in V. cholerae to the outer membrane secretin.     

Crystal structure of the N-terminal dimerisation domain of VicH, the H-NS-like protein of Vibrio cholerae

The histone-like nucleoid structuring (H-NS) protein is a global modulator of gene expression in Gram-negative bacteria. VicH, the H-NS protein of Vibrio cholerae, regulates the expression of certain major virulence determinants implicated in the pathogenesis of cholera. We present here the 2.5A crystal structure of the N-terminal oligomerisation domain of VicH (VicH_Nt). VicH_Nt adopts the same fold and dimeric assembly as the NMR structure of Escherichia coli H-NS_Nt, thus validating this fold against conflicting data. The structural similarity of V.cholerae VicH_Nt and E.coli H-NS_Nt, despite differences in origin, system of expression, experimental conditions and techniques used, indicates that the fold determined in our studies is robust to experimental conditions. Structural analysis and homology modelling were carried out to further elucidate the molecular basis of the functional polyvalence of the N-terminal domain. Our analysis of members of the H-NS superfamily supports the suggestion that the oligomerisation function of H-NS_Nt is conserved even in more distantly related proteins.     

Structural and functional studies on the interaction of GspC and GspD in the type II secretion system

Type II secretion systems (T2SSs) are critical for secretion of many proteins from Gram-negative bacteria. In the T2SS, the outer membrane secretin GspD forms a multimeric pore for translocation of secreted proteins. GspD and the inner membrane protein GspC interact with each other via periplasmic domains. Three different crystal structures of the homology region domain of GspC (GspC(HR)) in complex with either two or three domains of the N-terminal region of GspD from enterotoxigenic Escherichia coli show that GspC(HR) adopts an all-β topology. N-terminal β-strands of GspC and the N0 domain of GspD are major components of the interface between these inner and outer membrane proteins from the T2SS. The biological relevance of the observed GspC-GspD interface is shown by analysis of variant proteins in two-hybrid studies and by the effect of mutations in homologous genes on extracellular secretion and subcellular distribution of GspC in Vibrio cholerae. Substitutions of interface residues of GspD have a dramatic effect on the focal distribution of GspC in V. cholerae. These studies indicate that the GspC(HR)-GspD(N0) interactions observed in the crystal structure are essential for T2SS function. Possible implications of our structures for the stoichiometry of the T2SS and exoprotein secretion are discussed.     

Structure of the Pseudomonas aeruginosa XcpT pseudopilin,a major component of the type II secretion system

The bacterial type II protein secretion (T2S) and type IV piliation (T4P) systems share several common features. In particular, it is well established that the T2S system requires the function of a pilus-like structure, called pseudopilus, which is built upon assembly of pilin-like subunits, called pseudopilins. Pilins and pseudopilins have a hydrophobic N-terminal region, which precedes an extended hydrophilic C-terminal region. In the case of pilins, it was shown that oligomerisation and formation of helical fibers, takes place through interaction between the hydrophobic domains. XcpT, is the most abundant protein of the Pseudomonas aeruginosa T2S, and was proposed to be the main component in the pseudopilus. In this study we present the high-resolution NMR structure of the hydrophilic domain of XcpT (XcpTp). XcpTp is lacking the C-terminal disulfide bridged “D” domain found in type IV pilins and likely involved in receptor binding. This is in agreement with the idea that the XcpT-containing pseudopilus is required for protein secretion and not for bacterial attachment. Interestingly, by solving the 3D structure of XcpTp we revealed that the previously called αβ-loop pilin region is in fact highly conserved among major type II pseudopilins and constitutes a specific consensus motif for identifying major pseudopilins, which belong to this family.     

Domain requirements for DNA unwinding by mycobacterial UvrD2, an essential DNA helicase

Mycobacterial UvrD2 is a DNA-dependent ATPase with 3' to 5' helicase activity. UvrD2 is an atypical helicase, insofar as its N-terminal ATPase domain resembles the superfamily I helicases UvrD/PcrA, yet it has a C-terminal HRDC domain, which is a feature of RecQ-type superfamily II helicases. The ATPase and HRDC domains are connected by a CxxC-(14)-CxxC tetracysteine module that defines a new clade of UvrD2-like bacterial helicases found only in Actinomycetales. By characterizing truncated versions of Mycobacterium smegmatis UvrD2, we show that whereas the HRDC domain is not required for ATPase or helicase activities in vitro, deletion of the tetracysteine module abolishes duplex unwinding while preserving ATP hydrolysis. Replacing each of the CxxC motifs with a double-alanine variant AxxA had no effect on duplex unwinding, signifying that the domain module, not the cysteines, is crucial for function. The helicase activity of a truncated UvrD2 lacking the tetracysteine and HRDC domains was restored by the DNA-binding protein Ku, a component of the mycobacterial NHEJ system and a cofactor for DNA unwinding by the paralogous mycobacterial helicase UvrD1. Our findings indicate that coupling of ATP hydrolysis to duplex unwinding can be achieved by protein domains acting in cis or trans. Attempts to disrupt the M. smegmatis uvrD2 gene were unsuccessful unless a second copy of uvrD2 was present elsewhere in the chromosome, indicating that UvrD2 is essential for growth of M. smegmatis.     

New Insights into the Assembly of Bacterial Secretins: STRUCTURAL STUDIES OF THE PERIPLASMIC DOMAIN OF XcpQ FROM PSEUDOMONAS AERUGINOSA*

The type II secretion system is a multiprotein assembly spanning the inner and outer membranes in Gram-negative bacteria. It is found in almost all pathogenic bacteria where it contributes to virulence, host tissue colonization, and infection. The exoproteins are secreted across the outer membrane via a large translocation channel, the secretin, which typically adopts a dodecameric structure. These secretin channels have large periplasmic N-terminal domains that reach out into the periplasm for communication with the inner membrane platform and with a pseudopilus structure that spans the periplasm. Here we report the crystal structure of the N-terminal periplasmic domain of the secretin XcpQ from Pseudomonas aeruginosa, revealing a two-lobe dimeric assembly featuring parallel subunits engaging in well defined interactions at the tips of each lobe. We have employed structure-based engineering of disulfide bridges and native mass spectrometry to show that the periplasmic domain of XcpQ dimerizes in a concentration-dependent manner. Validation of these insights in the context of cellular full-length XcpQ and further evaluation of the functionality of disulfide-linked XcpQ establishes that the basic oligomerization unit of XcpQ is a dimer. This is consistent with the notion that the dodecameric secretin assembles as a hexamer of dimers to ensure correct projection of the N-terminal domains into the periplasm. Therefore, our studies provide a key conceptual advancement in understanding the assembly principles and dynamic function of type II secretion system secretins and challenge recent studies reporting monomers as the basic subunit of the secretin oligomer.     

Stepwise Folding of an Autotransporter Passenger Domain Is Not Essential for Its Secretion

Autotransporters are a superfamily of virulence proteins produced by Gram-negative bacteria. They consist of an N-terminal β-helical domain (“passenger domain”) that is secreted into the extracellular space and a C-terminal β-barrel domain (“β-domain”) that anchors the protein to the outer membrane. Because the periplasm lacks ATP, vectorial folding of the passenger domain in a C-to-N-terminal direction has been proposed to drive the secretion reaction. Consistent with this hypothesis, mutations that disrupt the folding of the C terminus of the passenger domain of the Escherichia coli O157:H7 autotransporter EspP have been shown to cause strong secretion defects. Here, we show that point mutations introduced at specific locations near the middle or N terminus of the EspP β-helix that perturb folding also impair secretion, but to a lesser degree. Surprisingly, we found that even multiple mutations that potentially abolish the stability of several consecutive rungs of the β-helix only moderately reduce secretion efficiency. Although these results provide evidence that the free energy derived from passenger domain folding contributes to secretion efficiency, they also suggest that a significant fraction of the energy required for secretion is derived from another source.