Small Molecule Antibiotics Inhibit Distinct Stages of Bacterial Outer Membrane Protein Assembly.

Several antibacterial compounds have recently been discovered that potentially inhibit the activity of BamA, an essential subunit of a heterooligomer (the barrel assembly machinery or BAM) that assembles outer membrane proteins (OMPs) in Gram-negative bacteria, but their mode of action is unclear. To address this issue, we examined the effect of three inhibitors on the biogenesis of a model E. coli OMP (EspP) in vivo. We found that darobactin potently inhibited the interaction of a conserved C-terminal sequence motif (the "ß signal") with BamA, but had no effect on assembly if added at a postbinding stage. In contrast, Polyphor peptide 7 and MRL-494 inhibited both binding and at least one later step of assembly. Taken together with previous studies that analyzed the binding of darobactin and Polyphor peptide 7 to BamA in vitro, our results strongly suggest that the two compounds inhibit BAM function by distinct competitive and allosteric mechanisms. In addition to providing insights into the properties of the antibacterial compounds, our results also provide direct experimental evidence that supports a model in which the binding of the ß signal to BamA initiates the membrane insertion of OMPs. IMPORTANCE There is a clear need to develop novel broad-spectrum antibiotics to address the global problem of antimicrobial resistance. Multiple compounds that have strong antibacterial activity have recently been described that appear to inhibit the activity of the barrel assembly machinery (BAM), an essential complex that catalyzes the assembly (i.e., folding and membrane insertion) of outer membrane proteins (OMPs) in all Gram-negative bacteria. We analyzed the effects of three of these compounds on OMP biogenesis in vivo and found that they inhibited different stages of the assembly process. Because these compounds have distinct modes of action, they can be used in combination to reduce the emergence of resistant strains. As a corollary, we obtained evidence that these compounds will be valuable tools in future studies on BAM function.

Reconstitution of Bam Complex-Mediated Assembly of a Trimeric Porin into Proteoliposomes.

Many integral membrane proteins form oligomeric complexes, but the assembly of these structures is poorly understood. Here, we show that the assembly of OmpC, a trimeric porin that resides in the Escherichia coli outer membrane (OM), can be reconstituted in vitro. Although we observed the insertion of both urea-denatured and in vitro-synthesized OmpC into pure lipid vesicles at physiological pH, the protein assembled only into dead-end dimers. In contrast, in vitro-synthesized OmpC was inserted into proteoliposomes that contained the barrel assembly machinery (Bam) complex, a conserved heterooligomer that catalyzes protein integration into the bacterial OM, and folded into heat-stable trimers by passing through a short-lived dimeric intermediate. Interestingly, complete OmpC assembly was also dependent on the addition of lipopolysaccharide (LPS), a glycolipid located exclusively in the OM. Our results strongly suggest that trimeric porins form through a stepwise process that requires the integration of the protein into the OM in an assembly-competent state. Furthermore, our results provide surprising evidence that interaction with LPS is required not only for trimerization but also for the productive insertion of individual subunits into the lipid bilayer. IMPORTANCE Porins are a widespread family of homotrimers that represent a substantial fraction of the total protein located in the OM of many Proteobacteria. These proteins facilitate the nonspecific diffusion of small molecules across the outer membrane and strongly influence the susceptibility of bacteria to clinically used antibiotics. The assembly of porins and the mechanism by which they are integrated into the outer membrane, however, are poorly understood. Here, we show that assembly can be completely reconstituted in vitro and requires only phospholipid vesicles containing the Bam complex, a molecular chaperone, and LPS. Furthermore, by showing that LPS binding is required for membrane insertion, our results demonstrate that a native lipid promotes a specific stage of porin biogenesis.

Bacterial Outer Membrane Proteins Are Targeted to the Bam Complex by Two Parallel Mechanisms.

Membrane proteins that are integrated into the outer membrane of Gram-negative bacteria typically contain a unique "ß barrel" structure that serves as a membrane spanning segment. A conserved "ß signal" motif is located at the C terminus of the ß barrel of many outer membrane proteins (OMPs), but the function of this sequence is unclear. We found that mutations in the ß signal slightly delayed the assembly of three model Escherichia coli OMPs by reducing their affinity for the barrel assembly machinery (Bam) complex, a heterooligomer that catalyzes ß barrel insertion, and led to the degradation of a fraction of the protein in the periplasm. Interestingly, the absence of the periplasmic chaperone SurA amplified the effect of the mutations and caused the complete degradation of the mutant proteins. In contrast, the absence of another periplasmic chaperone (Skp) suppressed the effect of the mutations and considerably enhanced the efficiency of assembly. Our results reveal the existence of two parallel OMP targeting mechanisms that rely on a cis-acting peptide (the ß signal) and a trans-acting factor (SurA), respectively. Our results also challenge the long-standing view that periplasmic chaperones are redundant and provide evidence that they have specialized functions.IMPORTANCE Proteins that are embedded in the outer membrane of Gram-negative bacteria (OMPs) play an important role in protecting the cell from harmful chemicals. OMPs share a common architecture and often contain a conserved sequence motif (ß motif) of unknown function. Although OMPs are escorted to the outer membrane by proteins called chaperones, the exact function of the chaperones is also unclear. Here, we show that the ß motif and the chaperone SurA both target OMPs to the ß barrel insertion machinery in the outer membrane. In contrast, the chaperone Skp delivers unintegrated OMPs to protein degradation complexes. Our results challenge the long-standing view that chaperones are functionally redundant and strongly suggest that they have specialized roles in OMP targeting and quality control.

Bam complex-mediated assembly of bacterial outer membrane proteins synthesized in an in vitro translation system.

Bacterial outer membrane proteins (OMPs) contain a unique "ß barrel" segment that is inserted into the membrane by the barrel assembly machinery (Bam) complex by an unknown mechanism. OMP assembly has been reconstituted in vitro, but assembly reactions have involved the use of urea-denatured protein purified from inclusion bodies. Here we show that the E. coli Bam complex catalyzes the efficient assembly of OMPs synthesized de novo in a coupled in vitro transcription/translation system. Interestingly, the in vitro translated forms of the OMPs we analyzed were assembled more rapidly and were effectively engaged by fewer periplasmic chaperones than their urea-denatured counterparts. Taken together, our results strongly suggest that the mode of production influences the conformational states sampled by OMPs and thereby affects their recognition by both chaperones and the Bam complex. Besides providing insights into OMP biogenesis, our work describes a novel, streamlined method to reconstitute OMP assembly in vitro.

Identification of a novel post-insertion step in the assembly of a bacterial outer membrane protein.

Although the barrel assembly machinery (Bam) complex has been shown to facilitate the insertion of ß barrel proteins into the bacterial outer membrane (OM), the stage at which ß barrels fold is unknown. Here, we describe insights into ß barrel assembly that emerged from an analysis of a member of the autotransporter family of OM proteins (EspP) in Escherichia coli. EspP contains an extracellular 'passenger' domain that is translocated across the OM and then released from the covalently linked ß barrel domain in an intra-barrel cleavage reaction. We found that the mutation of an unusual lipid-exposed lysine residue impairs a previously unidentified late folding step that follows both the membrane insertion of the ß barrel domain and the secretion of the passenger domain but that precedes proteolytic maturation. Our results demonstrate that ß barrel assembly can be completed at a post-insertion stage and raise the possibility that interactions with membrane lipids can promote folding in vivo. Furthermore, by showing that the passenger domain is secreted before the ß barrel domain is fully assembled, our results also provide evidence against the long-standing hypothesis that autotransporters are autonomous protein secretion systems.

Folding of a bacterial integral outer membrane protein is initiated in the periplasm.

The Bam complex promotes the insertion of ß-barrel proteins into the bacterial outer membrane, but it is unclear whether it threads ß-strands into the lipid bilayer in a stepwise fashion or catalyzes the insertion of pre-folded substrates. Here, to distinguish between these two possibilities, we analyze the biogenesis of UpaG, a trimeric autotransporter adhesin (TAA). TAAs consist of three identical subunits that together form a single ß-barrel domain and an extracellular coiled-coil ("passenger") domain. Using site-specific photocrosslinking to obtain spatial and temporal insights into UpaG assembly, we show that UpaG ß-barrel segments fold into a trimeric structure in the periplasm that persists until the termination of passenger-domain translocation. In addition to obtaining evidence that at least some ß-barrel proteins begin to fold before they interact with the Bam complex, we identify several discrete steps in the assembly of a poorly characterized class of virulence factors.

Selective pressure for rapid membrane integration constrains the sequence of bacterial outer membrane proteins.

Almost all bacterial outer membrane proteins (OMPs) contain a ß barrel domain that serves as a membrane anchor, but the assembly and quality control of these proteins are poorly understood. Here, we show that the introduction of a single lipid-facing arginine residue near the middle of the ß barrel of the Escherichia coli OMPs OmpLA and EspP creates an energy barrier that impedes membrane insertion. Although several unintegrated OmpLA mutants remained insertion-competent, they were slowly degraded by the periplasmic protease DegP. Two EspP mutants were also gradually degraded by DegP but were toxic because they first bound to the Bam complex, an essential heteroligomer that catalyzes the membrane insertion of OMPs. Interestingly, another EspP mutant likewise formed a prolonged, deleterious interaction with the Bam complex but was protected from degradation and eventually inserted into the membrane in a native conformation. The different types of interactions between the EspP mutants and the Bam complex that we observed may correspond to distinct stages in OMP assembly. Our results show that sequences that significantly delay assembly are disfavored not only because unintegrated OMPs are subjected to degradation, but also because OMPs that assemble slowly can form dominant-negative interactions with the Bam complex.

Reconstitution of bacterial autotransporter assembly using purified components.

Autotransporters are a superfamily of bacterial virulence factors consisting of an N-terminal extracellular ('passenger') domain and a C-terminal ß barrel ('ß') domain that resides in the outer membrane (OM). The mechanism by which the passenger domain is secreted is poorly understood. Here we show that a conserved OM protein insertase (the Bam complex) and a molecular chaperone (SurA) are both necessary and sufficient to promote the complete assembly of the Escherichia coli O157:H7 autotransporter EspP in vitro. Our results indicate that the membrane integration of the ß domain is the rate-limiting step in autotransporter assembly and that passenger domain translocation does not require the input of external energy. Furthermore, experiments using nanodiscs strongly suggest that autotransporter assembly is catalyzed by a single copy of the Bam complex. Finally, we describe a method to purify a highly active form of the Bam complex that should facilitate the elucidation of its function.

Mechanistic link between ß barrel assembly and the initiation of autotransporter secretion.

Autotransporters are bacterial virulence factors that contain an N-terminal extracellular ("passenger") domain and a C-terminal ß barrel ("ß") domain that anchors the protein to the outer membrane. The ß domain is required for passenger domain secretion, but its exact role in autotransporter biogenesis is unclear. Here we describe insights into the function of the ß domain that emerged from an analysis of mutations in the Escherichia coli O157:H7 autotransporter EspP. We found that the G1066A and G1081D mutations slightly distort the structure of the ß domain and delay the initiation of passenger domain translocation. Site-specific photocrosslinking experiments revealed that the mutations slow the insertion of the ß domain into the outer membrane, but do not delay the binding of the ß domain to the factor that mediates the insertion reaction (the Bam complex). Our results demonstrate that the ß domain does not simply target the passenger domain to the outer membrane, but promotes translocation when it reaches a specific stage of assembly. Furthermore, our results provide evidence that the Bam complex catalyzes the membrane integration of ß barrel proteins in a multistep process that can be perturbed by minor structural defects in client proteins.

Molecular basis for the activation of a catalytic asparagine residue in a self-cleaving bacterial autotransporter.

Autotransporters are secreted proteins produced by pathogenic Gram-negative bacteria. They consist of a membrane-embedded ß-domain and an extracellular passenger domain that is sometimes cleaved and released from the cell surface. We solved the structures of three noncleavable mutants of the autotransporter EspP to examine how it promotes asparagine cyclization to cleave its passenger. We found that cyclization is facilitated by multiple factors. The active-site asparagine is sterically constrained to conformations favorable for cyclization, while electrostatic interactions correctly orient the carboxamide group for nucleophilic attack. During molecular dynamics simulations, water molecules were observed to enter the active site and to form hydrogen bonds favorable for increasing the nucleophilicity of the active-site asparagine. When the activated asparagine attacks its main-chain carbonyl carbon, the resulting oxyanion is stabilized by a protonated glutamate. Upon cleavage, this proton could be transferred to the leaving amine group, helping overcome a significant energy barrier. Together, these findings provide insight into factors important for asparagine cyclization, a mechanism broadly used for protein cleavage.

Sequential and spatially restricted interactions of assembly factors with an autotransporter beta domain.

Autotransporters are bacterial virulence factors that consist of an N-terminal extracellular ("passenger") domain and a C-terminal ß barrel domain ("ß domain") that resides in the outer membrane. Here we used an in vivo site-specific photocrosslinking approach to gain insight into the mechanism by which the ß domain is integrated into the outer membrane and the relationship between ß domain assembly and passenger domain secretion. We found that periplasmic chaperones and specific components of the ß barrel assembly machinery (Bam) complex interact with the ß domain of the Escherichia coli O157:H7 autotransporter extracellular serine protease P (EspP) in a temporally and spatially regulated fashion. Although the chaperone Skp initially interacted with the entire ß domain, BamA, BamB, and BamD subsequently interacted with discrete ß domain regions. BamB and BamD remained bound to the ß domain longer than BamA and therefore appeared to function at a later stage of assembly. Interestingly, we obtained evidence that the completion of ß domain assembly is regulated by an intrinsic checkpoint mechanism that requires the completion of passenger domain secretion. In addition to leading to a detailed model of autotransporter biogenesis, our results suggest that the lipoprotein components of the Bam complex play a direct role in the membrane integration of ß barrel proteins.

The conformation of a nascent polypeptide inside the ribosome tunnel affects protein targeting and protein folding.

In this report, we describe insights into the function of the ribosome tunnel that were obtained through an analysis of an unusual 25 residue N-terminal motif (EspP(1-25) ) associated with the signal peptide of the Escherichia coli EspP protein. It was previously shown that EspP(1-25) inhibits signal peptide recognition by the signal recognition particle, and we now show that fusion of EspP(1-25) to a cytoplasmic protein causes it to aggregate. We obtained two lines of evidence that both of these effects are attributable to the conformation of EspP(1-25) inside the ribosome tunnel. First, we found that mutations in EspP(1-25) that abolished its effects on protein targeting and protein folding altered the cross-linking of short nascent chains to ribosomal components. Second, we found that a mutation in L22 that distorts the tunnel mimicked the effects of the EspP(1-25) mutations on protein biogenesis. Our results provide evidence that the conformation of a polypeptide inside the ribosome tunnel can influence protein folding under physiological conditions and suggest that ribosomal mutations might increase the solubility of at least some aggregation-prone proteins produced in E. coli.

Secretion of a bacterial virulence factor is driven by the folding of a C-terminal segment.

Autotransporters are bacterial virulence factors consisting of an N-terminal "passenger domain" that is secreted in a C- to-N-terminal direction and a C-terminal "ß domain" that resides in the outer membrane (OM). Although passenger domain secretion does not appear to use ATP, the energy source for this reaction is unknown. Here, we show that efficient secretion of the passenger domain of the Escherichia coli O157:H7 autotransporter EspP requires the stable folding of a C-terminal ≈17-kDa passenger domain segment. We found that mutations that perturb the folding of this segment do not affect its translocation across the OM but impair the secretion of the remainder of the passenger domain. Interestingly, an examination of kinetic folding mutants strongly suggested that the ≈17-kDa segment folds in the extracellular space. By mutagenizing the ≈17-kDa segment, we also fortuitously isolated a unique translocation intermediate. Analysis of this intermediate suggests that a heterooligomer that facilitates the membrane integration of OM proteins (the Bam complex) also promotes the surface exposure of the ≈17-kDa segment. Our results provide direct evidence that protein folding can drive translocation and help to clarify the mechanism of autotransporter secretion.

An unusual signal peptide extension inhibits the binding of bacterial presecretory proteins to the signal recognition particle, trigger factor, and the SecYEG complex.

Considerable evidence indicates that the Escherichia coli signal recognition particle (SRP) selectively targets proteins that contain highly hydrophobic signal peptides to the SecYEG complex cotranslationally. Presecretory proteins that contain only moderately hydrophobic signal peptides typically interact with trigger factor (TF) and are targeted post-translationally. Here we describe a striking exception to this rule that has emerged from the analysis of an unusual 55-amino acid signal peptide associated with the E. coli autotransporter EspP. The EspP signal peptide consists of a C-terminal domain that resembles a classical signal peptide plus an N-terminal extension that is conserved in other autotransporter signal peptides. Although a previous study showed that proteins containing the C-terminal domain of the EspP signal peptide are targeted cotranslationally by SRP, we found that proteins containing the full-length signal peptide were targeted post-translationally via a novel TF-independent mechanism. Mutation of an invariant asparagine residue in the N-terminal extension, however, restored cotranslational targeting. Remarkably, proteins containing extremely hydrophobic derivatives of the EspP signal peptide were also targeted post-translationally. These and other results suggest that the N-terminal extension alters the accessibility of the signal peptide to SRP and TF and promotes post-translational export by reducing the efficiency of the interaction between the signal peptide and the SecYEG complex. Based on data, we propose that the N-terminal extension mediates an interaction with an unidentified cytoplasmic factor or induces the formation of an unusual signal peptide conformation prior to the onset of protein translocation.

Efficient secretion of a folded protein domain by a monomeric bacterial autotransporter.

Bacterial autotransporters are proteins that contain a small C-terminal 'beta domain' that facilitates translocation of a large N-terminal 'passenger domain' across the outer membrane (OM) by an unknown mechanism. Here we used EspP, an autotransporter produced by Escherichia coli 0157:H7, as a model protein to gain insight into the transport reaction. Initially we found that the passenger domain of a truncated version of EspP (EspPDelta1-851) was translocated efficiently across the OM. Blue Native polyacrylamide gel electrophoresis, analytical ultracentrifugation and other biochemical methods showed that EspPDelta1-851 behaves as a compact monomer and strongly suggest that the channel formed by the beta domain is too narrow to accommodate folded polypeptides. Surprisingly, we found that a folded protein domain fused to the N-terminus of EspPDelta1-851 was efficiently translocated across the OM. Further analysis revealed that the passenger domain of wild-type EspP also folds at least partially in the periplasm. To reconcile these data, we propose that the EspP beta domain functions primarily to target and anchor the protein and that an external factor transports the passenger domain across the OM.

An unusual signal peptide facilitates late steps in the biogenesis of a bacterial autotransporter.

Bacterial autotransporters are proteins that use a C-terminal porin-like domain to facilitate the transport of an upstream "passenger domain" across the outer membrane. Although autotransporters are translocated across the inner membrane (IM) via the Sec pathway, some of them contain exceptionally long signal peptides distinguished by a unique N-terminal sequence motif. In this study, we used the Escherichia coli O157:H7 autotransporter EspP as a model protein to investigate the function of the unusual signal peptides. We found that removal of the N-terminal motif or replacement of the EspP signal peptide did not affect translocation of the protein across the IM. Remarkably, modification of the signal peptide caused EspP to misfold in the periplasm and blocked transport of the passenger domain across the outer membrane. Further analysis suggested that the EspP signal peptide transits slowly through the Sec machinery. Based on these results, we propose that the unusual signal peptides not only function as targeting signals, but also prevent misfolding of the passenger domain in the periplasm by transiently tethering it to the IM.

Basic amino acids in a distinct subset of signal peptides promote interaction with the signal recognition particle.

Previous studies have demonstrated that signal peptides bind to the signal recognition particle (SRP) primarily via hydrophobic interactions with the 54-kDa protein subunit. The crystal structure of the conserved SRP ribonucleoprotein core, however, raised the surprising possibility that electrostatic interactions between basic amino acids in signal peptides and the phosphate backbone of SRP RNA may also play a role in signal sequence recognition. To test this possibility we examined the degree to which basic amino acids in a signal peptide influence the targeting of two Escherichia coli proteins, maltose binding protein and OmpA. Whereas both proteins are normally targeted to the inner membrane by SecB, we found that replacement of their native signal peptides with another moderately hydrophobic but unusually basic signal peptide (DeltaEspP) rerouted them into the SRP pathway. Reduction in either the net positive charge or the hydrophobicity of the DeltaEspP signal peptide decreased the effectiveness of SRP recognition. A high degree of hydrophobicity, however, compensated for the loss of basic residues and restored SRP binding. Taken together, the data suggest that the formation of salt bridges between SRP RNA and basic amino acids facilitates the binding of a distinct subset of signal peptides whose hydrophobicity falls slightly below a threshold level.