Harris D. Bernstein

The E. coli Signal Recognition Particle Is Required for the Insertion of a Subset of Inner Membrane Proteins

E. coli homologs of the signal recognition particle (SRP) and its receptor are essential for viability, but their role in protein export is unclear. To elucidate their function, we devised a genome-wide screen to identify genes that encode SRP substrates. Inhibition of the SRP pathway sharply blocked the membrane insertion of several polytopic inner membrane proteins (IMPs) that were predicted to be SRP substrates, but had a smaller effect on the insertion of other IMPs and no significant effect on preprotein translocation. Our results suggest that whereas most E. coli preproteins and some IMPs can utilize SRP-independent targeting pathways effectively, the structural features of a subset of IMPs have required the conservation of an SRP-based targeting machinery.

The methionine-rich domain of the 54 kd protein subunit of the signal recognition particle contains an RNA binding site and can be crosslinked to a signal sequence.

The 54 kd protein subunit of the signal recognition particle (SRP54) has been shown to bind signal sequences by UV crosslinking. Primary structure analysis and phylogenetic comparisons have suggested that SRP54 is composed of two domains: an amino‐terminal domain that contains a putative GTP‐binding site (G‐domain) and a carboxy‐terminal domain that contains a high abundance of methionine residues (M‐domain). Partial proteolysis of SRP revealed that the two proposed domains of SRP54 indeed represent structurally discrete entities. Upon proteolysis the intact G‐domain was released from SRP, whereas the M‐domain remained attached to the core of the particle. Reconstitution experiments demonstrated that the isolated M‐domain associates with 7SL RNA in the presence of SRP19. In addition, we observed a specific binding of the M‐domain directly to 4.5S RNA of Escherichia coli, which contains a structural motif also present in 7SL RNA. This shows that the M‐domain contains an RNA binding site, and suggests that SRP54 may be linked to the rest of SRP through this domain by a direct interaction with 7SL RNA. Using UV crosslinking, we found that in an in vitro translation system the preprolactin signal sequence contacts SRP through the M‐domain of SRP54. These results imply that the M‐domain contains the signal sequence binding site of SRP54, although we cannot exclude that the G‐domain may also be in proximity to bound signal sequences.(ABSTRACT TRUNCATED AT 250 WORDS)

The targeting pathway of Escherichia coli presecretory and integral membrane proteins is specified by the hydrophobicity of the targeting signal

Previous studies have demonstrated that presecretory proteins such as maltose binding protein (MBP) and outer membrane protein A (OmpA) are targeted to the Escherichia coli inner membrane by the molecular chaperone SecB, but that integral membrane proteins are targeted by the signal recognition particle (SRP). In vitro studies have suggested that trigger factor binds to a sequence near the N terminus of the mature region of OmpA and shunts the protein into the SecB pathway by blocking an interaction between SRP and the signal peptide. By contrast, we have found that the targeting pathway of a protein under physiological conditions is dictated by the composition of its targeting signal. Replacement of the MBP or OmpA signal peptide with the first transmembrane segment of AcrB abolished the dependence on SecB for transport and rerouted both proteins into the SRP targeting pathway. More modest alterations of the MBP signal peptide that simply increase its hydrophobicity also promoted SRP binding. Furthermore, we obtained evidence that SRP has a low affinity for typical signal peptides in vivo. These results imply that different classes of E. coli proteins are targeted by distinct pathways because bacterial SRP binds to a more restricted range of targeting signals than its eukaryotic counterpart.

Interaction of an autotransporter passenger domain with BamA during its translocation across the bacterial outer membrane

Autotransporters are a superfamily of virulence factors produced by Gram-negative bacteria consisting of a large N-terminal extracellular domain (“passenger domain”) and a C-terminal β barrel domain (“β domain”). The mechanism by which the passenger domain is translocated across the outer membrane (OM) is unknown. Here we show that the insertion of a small linker into the passenger domain of the Escherichia coli O157:H7 autotransporter EspP effectively creates a translocation intermediate by transiently stalling translocation near the site of the insertion. Using a site-specific photocrosslinking approach, we found that residues adjacent to the stall point interact with BamA, a component of a heterooligomeric complex (Bam complex) that catalyzes OM protein assembly, and that residues closer to the EspP N terminus interact with the periplasmic chaperones SurA and Skp. The EspP–BamA interaction was short-lived and could be detected only when passenger domain translocation was stalled. These results support a model in which molecular chaperones prevent misfolding of the passenger domain before its secretion and the Bam complex catalyzes both the integration of the β domain into the OM and the translocation of the passenger domain across the OM in a C- to N-terminal direction.

Autotransporter structure reveals intra-barrel cleavage followed by conformational changes.

Autotransporters are virulence factors produced by Gram-negative bacteria. They consist of two domains, an N-terminal passenger domain and a C-terminal β-domain. β-domains form β-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-Å structure of the post-cleavage state of the β-domain of EspP, an autotransporter produced by Escherichia coli strain O157:H7. The structure consists of a 12-stranded β-barrel with the passenger domain–β-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 β-domain after cleavage, one conferring increased stability on the β-domain and another restricting access to the barrel pore.

Signal Recognition Particle (SRP)-mediated Targeting and Sec-dependent Translocation of an Extracellular Escherichia coli Protein

Hemoglobin protease (Hbp) is a hemoglobin-degrading protein that is secreted by a human pathogenicEscherichia coli strain via the autotransporter mechanism. Little is known about the earliest steps in autotransporter secretion,i.e. the targeting to and translocation across the inner membrane. Here, we present evidence that Hbp interacts with the signal recognition particle (SRP) and the Sec-translocon early during biogenesis. Furthermore, Hbp requires a functional SRP targeting pathway and Sec-translocon for optimal translocation across the inner membrane. SecB is not required for targeting of Hbp but can compensate to some extent for the lack of SRP. Hbp is synthesized with an unusually long signal peptide that is remarkably conserved among a subset of autotransporters. We propose that these autotransporters preferentially use the co-translational SRP/Sec route to avoid adverse effects of the exposure of their mature domains in the cytoplasm.

Cleavage of a bacterial autotransporter by an evolutionarily convergent autocatalytic mechanism

Bacterial autotransporters are comprised of an N‐terminal ‘passenger domain’ and a C‐terminal β barrel (‘β domain’) that facilitates transport of the passenger domain across the outer membrane. Following translocation, the passenger domains of some autotransporters are cleaved by an unknown mechanism. Here we show that the passenger domain of the Escherichia coli O157:H7 autotransporter EspP is released in a novel autoproteolytic reaction. After purification, the uncleaved EspP precursor underwent proteolytic processing in vitro. An analysis of protein topology together with mutational studies strongly suggested that the reaction occurs inside the β barrel and revealed that two conserved residues, an aspartate within the β domain (Asp1120) and an asparagine (Asn1023) at the P1 position of the cleavage junction, are essential for passenger domain cleavage. Interestingly, these residues were also essential for the proteolytic processing of two distantly related autotransporters. The data strongly suggest that Asp1120 and Asn1023 form an unusual catalytic dyad that mediates self‐cleavage through the cyclization of the asparagine. Remarkably, a very similar mechanism has been proposed for the maturation of eukaryotic viral capsids.

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

Bacterial autotransporters are proteins that contain a small C‐terminal ‘β 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 (EspPΔ1‐851) was translocated efficiently across the OM. Blue Native polyacrylamide gel electrophoresis, analytical ultracentrifugation and other biochemical methods showed that EspPΔ1‐851 behaves as a compact monomer and strongly suggest that the channel formed by the β domain is too narrow to accommodate folded polypeptides. Surprisingly, we found that a folded protein domain fused to the N‐terminus of EspPΔ1‐851 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 β domain functions primarily to target and anchor the protein and that an external factor transports the passenger domain across the OM.

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

Autotransporters are bacterial virulence factors that consist of an N-terminal extracellular (“passenger”) domain and a C-terminal β barrel domain (“β 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 β domain is integrated into the outer membrane and the relationship between β domain assembly and passenger domain secretion. We found that periplasmic chaperones and specific components of the β barrel assembly machinery (Bam) complex interact with the β 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 β domain, BamA, BamB, and BamD subsequently interacted with discrete β domain regions. BamB and BamD remained bound to the β domain longer than BamA and therefore appeared to function at a later stage of assembly. Interestingly, we obtained evidence that the completion of β 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 β barrel proteins.

The Plasticity of a Translation Arrest Motif Yields Insights into Nascent Polypeptide Recognition inside the Ribosome Tunnel

The recognition of a C-terminal motif in E. coli SecM ((150)FXXXXWIXXXXGIRAGP(166)) inside the ribosome tunnel causes translation arrest, but the mechanism of recognition is unknown. Whereas single mutations in this motif impair recognition, we demonstrate that new arrest-inducing peptides can be created through remodeling of the SecM C terminus. We found that R163 is indispensable but that flanking residues that vary in number and position play an important secondary role in translation arrest. The observation that individual SecM variants showed a distinct pattern of crosslinking to ribosomal proteins suggests that each peptide adopts a unique conformation inside the tunnel. Based on the results, we propose that translation arrest occurs when the peptide conformation specified by flanking residues moves R163 into a precise intratunnel location. Our data indicate that translation arrest results from extensive communication between SecM and the tunnel and help to explain the striking diversity of arrest-inducing peptides found throughout nature.