Molecular architecture and function of the Omp85 family of proteins

Omp85 is a protein found in Gram-negative bacteria where it serves to integrate proteins into the bacterial outer membrane. Members of the Omp85 family of proteins are defined by the presence of two domains: an N-terminal, periplasmic domain rich in POTRA repeats and a C-terminal beta-barrel domain embedded in the outer membrane. The widespread distribution of Omp85 family members together with their fundamental role in outer membrane assembly suggests the ancestral Omp85 arose early in the evolution of prokaryotic cells. Mitochondria, derived from an ancestral bacterial endosymbiont, also use a member of the Omp85 family to assemble proteins in their outer membranes. More distant relationships are seen between the Omp85 family and both the core proteins in two-partner secretion systems and the Toc75 family of protein translocases found in plastid outer envelopes. Aspects of the ancestry and molecular architecture of the Omp85 family of proteins is providing insight into the mechanism by which proteins might be integrated and assembled into bacterial outer membranes.     

Chloroplast outer envelope protein P39 in Arabidopsis thaliana belongs to the Omp85 protein family

Proteins of the Omp85 family chaperone the membrane insertion of β‐barrel‐shaped outer membrane proteins in bacteria, mitochondria, and probably chloroplasts and facilitate the transfer of nuclear‐encoded cytosolically synthesized preproteins across the outer envelope of chloroplasts. This protein family is characterized by N‐terminal polypeptide transport‐associated (POTRA) domains and a C‐terminal membrane‐embedded β‐barrel. We have investigated a recently identified Omp85 family member of Arabidopsis thaliana annotated as P39. We show by in vitro and in vivo experiments that P39 is localized in chloroplasts. The electrophysiological properties of P39 are consistent with those of other Omp85 family members confirming the sequence based assignment of P39 to this family. Bioinformatic analysis showed that P39 lacks any POTRA domain, while a complete 16 stranded β‐barrel including the highly conserved L6 loop is proposed. The electrophysiological properties are most comparable to Toc75‐V, which is consistent with the phylogenetic clustering of P39 in the Toc75‐V rather than the Toc75‐III branch of the Omp85 family tree. Taken together P39 forms a pore with Omp85 family protein characteristics. The bioinformatic comparison of the pore region of Toc75‐III, Toc75‐V, and P39 shows distinctions of the barrel region most likely related to function. Proteins 2017; 85:1391–1401. © 2014 Wiley Periodicals, Inc.     

The outer membrane Omp85‐like protein P39 influences metabolic homeostasis in mature Arabidopsis thaliana

• The Omp85 proteins form a large membrane protein family in bacteria and eukaryotes. Omp85 proteins are composed of a C‐terminal β‐barrel‐shaped membrane domain and one or more N‐terminal polypeptide transport‐associated (POTRA) domains. However, Arabidopsis thaliana contains two genes coding for Omp85 proteins without a POTRA domain. One gene is designated P39, according to the molecular weight of the encoded protein. The protein is targeted to plastids and it was established that p39 has electrophysiological properties similar to other Omp85 family members, particularly to that designated as Toc75V/Oep80.
• We analysed expression of the gene and characterised two T‐DNA insertion mutants, focusing on alterations in photosynthetic activity, plastid ultrastructure, global expression profile and metabolome.
• We observed pronounced expression of P39, especially in veins. Mutants of P39 show growth aberrations, reduced photosynthetic activity and changes in plastid ultrastructure, particularly in the leaf tip. Further, they display global alteration of gene expression and metabolite content in leaves of mature plants.
• We conclude that the function of the plastid‐localised and vein‐specific Omp85 family protein p39 is important, but not essential, for maintenance of metabolic homeostasis of full‐grown A. thaliana plants. Further, the function of p39 in veins influences the functionality of other plant tissues. The link connecting p39 function with metabolic regulation in mature A. thaliana is discussed.

     

First glimpse of the crystal structure of YaeT's POTRA domains.

The Omp85/YaeT family of proteins, which are conserved from bacteria to human, catalyzes insertion and assembly of proteins in the outer membrane. The structure consists of a transmembrane beta-barrel domain and a soluble polypeptide-transport-associated (POTRA) domain. The POTRA domain is critical for substrate recognition and perhaps substrate folding, while the beta-barrel domain assists in membrane insertion. The resolution of the crystal structure of the POTRA domain of the Escherichia coli YaeT protein provides a possible molecular mechanism by which the diverse group of substrates is recognized. Knowledge gained from the crystal structure may also spur the development of a novel class of chemotherapeutic inhibitors.     

Omp85 from the Thermophilic Cyanobacterium Thermosynechococcus elongatus Differs from Proteobacterial Omp85 in Structure and Domain Composition

Omp85 proteins are essential proteins located in the bacterial outer membrane. They are involved in outer membrane biogenesis and assist outer membrane protein insertion and folding by an unknown mechanism. Homologous proteins exist in eukaryotes, where they mediate outer membrane assembly in organelles of endosymbiotic origin, the mitochondria and chloroplasts. We set out to explore the homologous relationship between cyanobacteria and chloroplasts, studying the Omp85 protein from the thermophilic cyanobacterium Thermosynechococcus elongatus. Using state-of-the art sequence analysis and clustering methods, we show how this protein is more closely related to its chloroplast homologue Toc75 than to proteobacterial Omp85, a finding supported by single channel conductance measurements. We have solved the structure of the periplasmic part of the protein to 1.97 Å resolution, and we demonstrate that in contrast to Omp85 from Escherichia coli the protein has only three, not five, polypeptide transport-associated (POTRA) domains, which recognize substrates and generally interact with other proteins in bigger complexes. We model how these POTRA domains are attached to the outer membrane, based on the relationship of Omp85 to two-partner secretion system proteins, which we show and analyze. Finally, we discuss how Omp85 proteins with different numbers of POTRA domains evolved, and evolve to this day, to accomplish an increasing number of interactions with substrates and helper proteins.     

Dynamic interplay of membrane‐proximal POTRA domain and conserved loop L6 in Omp85 transporter FhaC

Omp85 transporters mediate protein insertion into, or translocation across, membranes. They have a conserved architecture, with POTRA domains that interact with substrate proteins, a 16‐stranded transmembrane β barrel, and an extracellular loop, L6, folded back in the barrel pore. Here using electrophysiology, in vivo biochemical approaches and electron paramagnetic resonance, we show that the L6 loop of the Omp85 transporter FhaC changes conformation and modulates channel opening. Those conformational changes involve breaking the conserved interaction between the tip of L6 and the inner β‐barrel wall. The membrane‐proximal POTRA domain also exchanges between several conformations, and the binding of FHA displaces this equilibrium. We further demonstrate a dynamic, physical communication between the POTRA domains and L6, which must take place via the β barrel. Our findings thus link all three essential components of Omp85 transporters and indicate that they operate in a concerted fashion in the transport cycle.     

Tic22 from Anabaena sp. PCC 7120 with holdase function involved in outer membrane protein biogenesis shuttles between plasma membrane and Omp85

β‐barrel‐shaped outer membrane proteins (OMPs) ensure regulated exchange of molecules across the cell‐wall of Gram‐negative bacteria. They are synthesized in the cytoplasm and translocated across the plasma membrane via the SEC translocon. In the periplasm, several proteins participate in the transfer of OMPs to the outer membrane‐localized complex catalyzing their insertion. This process has been described in detail for proteobacteria and some molecular components are conserved in cyanobacteria. For example, Omp85 proteins that catalyze the insertion of OMPs into the outer membrane exist in cyanobacteria as well. In turn, SurA and Skp involved in OMP transfer from plasma membrane to Omp85 in E. coli are likely replaced by Tic22 in cyanobacteria. We describe that anaTic22 functions as periplasmic holdase for OMPs in Anabaena sp. PCC 7120 and provide evidence for the process of substrate delivery to anaOmp85. AnaTic22 binds to the plasma membrane with specificity for phosphatidylglycerol and monogalactosyldiacylglycerol. Substrate recognition induces membrane dissociation and interaction with the N‐terminal POTRA domain of Omp85. This leads to substrate release by the interaction with a proline‐rich domain and the first POTRA domain of Omp85. The order of events during OMP transfer from plasma membrane to Omp85 in cyanobacteria is discussed.     

Fold and function of polypeptide transport-associated domains responsible for delivering unfolded proteins to membranes

Membranes of Gram-negative bacteria, mitochondria and chloroplasts receive and fold beta-barrel transmembrane proteins through the action of polypeptide transport-associated (POTRA) domains. In Escherichia coli, folding substrates are inserted into the outer membrane by the essential protein YaeT, a prototypic Omp85 protein. Here, the articulation between tandem POTRA domains in solution is defined by nuclear magnetic resonance (NMR) spectroscopy, indicating an unprecedented juxtaposition. The novel solution orientations of all five POTRA domains are revealed by small-angle X-ray scattering of the entire 46 kDa periplasmic region. NMR titration studies show that strands from YaeT's canonical folding substrate, PhoE, bind non-specifically along alternating sides of its mixed beta sheets, thus providing an ideal platform for helping to fold nascent outer-membrane proteins. Together, this provides the first structural model of how multiple POTRA domains recruit substrates from the periplasmic solution into the outer membrane.     

Conserved Properties of Polypeptide Transport-associated (POTRA) Domains Derived from Cyanobacterial Omp85

Proteins of the Omp85 family are conserved in all kingdoms of life. They mediate protein transport across or protein insertion into membranes and reside in the outer membranes of Gram-negative bacteria, mitochondria, and chloroplasts. Omp85 proteins contain a C-terminal transmembrane β-barrel and a soluble N terminus with a varying number of polypeptide-transport-associated or POTRA domains. Here we investigate Omp85 from the cyanobacterium Anabaena sp. PCC 7120. The crystallographic three-dimensional structure of the N-terminal region shows three POTRA domains, here named P1 to P3 from the N terminus. Molecular dynamics simulations revealed a hinge between P1 and P2 but in contrast show that P2 and P3 are fixed in orientation. The P2-P3 arrangement is identical as seen for the POTRA domains from proteobacterial FhaC, suggesting this orientation is a conserved feature. Furthermore, we define interfaces for protein-protein interaction in P1 and P2. P3 possesses an extended loop unique to cyanobacteria and plantae, which influences pore properties as shown by deletion. It now becomes clear how variations in structure of individual POTRA domains, as well as the different number of POTRA domains with both rigid and flexible connections make the N termini of Omp85 proteins versatile adaptors for a plentitude of functions.     

Requirements for the import of neisserial Omp85 into the outer membrane of human mitochondria

β-Barrel proteins are present only in the outer membranes of Gram-negative bacteria, chloroplasts and mitochondria. Fungal mitochondria were shown to readily import and assemble bacterial β-barrel proteins, but human mitochondria exhibit certain selectivity. Whereas enterobacterial β-barrel proteins are not imported, neisserial ones are. Of those, solely neisserial Omp85 is integrated into the outer membrane of mitochondria. In this study, we wanted to identify the signal that targets neisserial β-barrel proteins to mitochondria. We exchanged parts of neisserial Omp85 and PorB with their Escherichia coli homologues BamA and OmpC. For PorB, we could show that its C-terminal quarter can direct OmpC to mitochondria. In the case of Omp85, we could identify several amino acids of the C-terminal β-sorting signal as crucial for mitochondrial targeting. Additionally, we found that at least two POTRA (polypeptide-transport associated) domains and not only the β-sorting signal of Omp85 are needed for its membrane integration and function in human mitochondria. We conclude that the signal that directs neisserial β-barrel proteins to mitochondria is not conserved between these proteins. Furthermore, a linear mitochondrial targeting signal probably does not exist. It is possible that the secondary structure of β-barrel proteins plays a role in directing these proteins to mitochondria.     

Secondary structure and 1H, 13C and 15N resonance assignments of the Escherichia coli YaeT POTRA domain

We report the first ¹H, ¹³C and ¹⁵N chemical shift assignments and secondary structure of the Escherichia coli YaeT POTRA domain; a domain found in the Omp85 family of proteins which is critical for insertion and folding of outer membrane proteins in Gram-negative bacteria.     

Conserved Residues of the Putative L6 Loop of Escherichia coli BamA Play a Critical Role in the Assembly of β-Barrel Outer Membrane Proteins, Including That of BamA Itself

Many members of the Omp85 family of proteins form essential β-barrel outer membrane protein (OMP) biogenesis machinery in Gram-negative bacteria, chloroplasts, and mitochondria. In Escherichia coli, BamA, a member of the Omp85 family, folds into an outer membrane-embedded β-barrel domain and a soluble periplasmic polypeptide-transport-associated (POTRA) domain. Although the high-resolution structures of only the BamA POTRA domain of E. coli are available, the crystal structure of FhaC, an Omp85 family member and a component of the two-partner secretion system in Bordetella pertussis, suggests that the BamA β-barrel likely folds into a 16-stranded β-barrel. The FhaC β-barrel is occluded by an N-terminal α-helix and a large β-barrel loop, L6, which carries residues that are highly conserved among the Omp85 family members. Deletion of L6 in FhaC did not affect its biogenesis but abolished its secretion function. In this study, we tested the hypothesis that the conserved residues of the putative L6 loop, which presumably folds back into the lumen of the BamA β-barrel like the FhaC counterpart, play an important role in OMP and/or BamA biogenesis. The conserved (641)RGF(643) residues of L6 were either deleted or replaced with alanine in various permutations. Phenotypic and biochemical characterization of various BamA L6 mutants revealed that the conserved RGF residues are critical for OMP biogenesis. Moreover, three BamA L6 alterations, ΔRGF, AAA, and AGA, produced a conditional lethal phenotype, concomitant with severely reduced BamA levels and folding defects. Thus, the conserved (641)RGF(643) residues of the BamA L6 loop are important for BamA folding and biogenesis.     

Biogenesis of mitochondrial β-barrel proteins: the POTRA domain is involved in precursor release from the SAM complex

The mitochondrial outer membrane contains proteinaceous machineries for the translocation of precursor proteins. The sorting and assembly machinery (SAM) is required for the insertion of β-barrel proteins into the outer membrane. Sam50 is the channel-forming core subunit of the SAM complex and belongs to the BamA/Sam50/Toc75 family of proteins that have been conserved from Gram-negative bacteria to mitochondria and chloroplasts. These proteins contain one or more N-terminal polypeptide transport-associated (POTRA) domains. POTRA domains can bind precursor proteins, however, different views exist on the role of POTRA domains in the biogenesis of β-barrel proteins. It has been suggested that the single POTRA domain of mitochondrial Sam50 plays a receptor-like function at the SAM complex. We established a system to monitor the interaction of chemical amounts of β-barrel precursor proteins with the SAM complex of wild-type and mutant yeast in organello. We report that the SAM complex lacking the POTRA domain of Sam50 efficiently binds β-barrel precursors, but is impaired in the release of the precursors. These results indicate the POTRA domain of Sam50 is not essential for recognition of β-barrel precursors but functions in a subsequent step to promote the release of precursor proteins from the SAM complex.     

Characterization of pores formed by YaeT (Omp85) from Escherichia coli

Proteins of the Omp85 family play a major role in the biogenesis of the bacterial outer membrane, since they were shown to mediate insertion of outer membrane proteins. The Escherichia coli Omp85 homologue YaeT is essential for viability, but its exact mode of action is not yet elucidated. We could show that YaeT is composed of two distinct domains, an amino-terminal periplasmic and a carboxy-terminal membrane domain. The full length YaeT and the isolated membrane domain induce pores when reconstituted in planar lipid membranes. The pores exhibit a certain variability of conductance indicating a flexible structure, which could be an essential property of a lateral opening channel releasing proteins into the bacterial outer membrane. We could further show that the periplasmic domain proves to be essential for in vivo function of YaeT.     

Cross‐species chimeras reveal BamA POTRA and β‐barrel domains must be fine‐tuned for efficient OMP insertion

BAM is a conserved molecular machine, the central component of which is BamA. Orthologues of BamA are found in all Gram‐negative bacteria, chloroplasts and mitochondria where it is required for the folding and insertion of β‐barrel containing integral outer membrane proteins (OMPs) into the outer membrane. BamA binds unfolded β‐barrel precursors via the five polypeptide transport‐associated (POTRA) domains at its N‐terminus. The C‐terminus of BamA folds into a β‐barrel domain, which tethers BamA to the outer membrane and is involved in OMP insertion. BamA orthologues are found in all Gram‐negative bacteria and appear to function in a species‐specific manner. Here we investigate the nature of this species‐specificity by examining whether chimeric Escherichia coli BamA fusion proteins, carrying either the β‐barrel or POTRA domains from various BamA orthologues, can functionally replace E. coli BamA. We demonstrate that the β‐barrel domains of many BamA orthologues are functionally interchangeable. We show that defects in the orthologous POTRA domains can be rescued by compensatory mutations within the β‐barrel. These data reveal that the POTRA and barrel domains must be precisely aligned to ensure efficient OMP insertion.     

The Polypeptide Transport-associated (POTRA) Domains of TpsB Transporters Determine the System Specificity of Two-partner Secretion Systems

The two-partner secretion (TPS) systems of Gram-negative bacteria secrete large TpsA exoproteins by a dedicated TpsB transporter in the outer membrane. TpsBs contain an N-terminal module located in the periplasm that includes two polypeptide transport-associated (POTRA) domains. These are thought to initiate secretion of a TpsA by binding its N-terminal secretion signal, called the TPS domain. Neisseria meningitidis encodes up to five TpsA proteins that are secreted via only two TpsB transporters: TpsB1 and TpsB2. Of these two, the TpsB2 recognizes the TPS domains of all TpsAs, despite their sequence diversity. By contrast, the TpsB1 shows a limited recognition of a TPS domain that is shared by two TpsAs. The difference in substrate specificity of the TpsBs enabled us to investigate the role of the POTRA domains in the selection of TPS domains. We tested secretion of TPS domains or full-length TpsAs by TpsB mutants with deleted, duplicated, and exchanged POTRA domains. Exchanging the two POTRA domains of a TpsB resulted in a switch in specificity. Furthermore, exchanging a single POTRA domain showed that each of the two domains contributed to the cargo selection. Remarkably, the order of the POTRA domains could be reversed without affecting substrate selection, but this aberrant order did result in an alternatively processed secretion product. Our results suggest that secretion of a TpsA is initiated by engaging both POTRA domains of a TpsB transporter and that these select the cognate TpsAs for secretion.     

Solid-State NMR Studies of Full-Length BamA in Lipid Bilayers Suggest Limited Overall POTRA Mobility

The outer membrane protein BamA is the key player in β-barrel assembly in Gram-negative bacteria. Despite the availability of high-resolution crystal structures, the dynamic behavior of the transmembrane domain and the large periplasmic extension consisting of five POTRA (POlypeptide-TRansport-Associated) domains remains unclear. We demonstrate reconstitution of full-length BamA in proteoliposomes at low lipid-to-protein ratio, leading to high sensitivity and resolution in solid-state NMR (ssNMR) experiments. We detect POTRA domains in ssNMR experiments probing rigid protein segments in our preparations. These results suggest that the periplasmic region of BamA is firmly attached to the β-barrel and does not experience fast global motion around the angle between POTRA 2 and 3. We show that this behavior holds at lower protein concentrations and elevated temperatures. Chemical shift variations observed after reconstitution in lipids with different chain lengths and saturation levels are compatible with conformational plasticity of BamA's transmembrane domain. Electron microscopy of the ssNMR samples shows that BamA can cause local disruptions of the lipid bilayer in proteoliposomes. The observed interplay between protein–protein and protein–lipid interactions may be critical for BamA-mediated insertion of substrates into the outer membrane.     

Dissection of β‐barrel outer membrane protein assembly pathways through characterizing BamA POTRA 1 mutants of Escherichia coli

BamA of Escherichia coli is an essential component of the hetero‐oligomeric machinery that mediates β‐barrel outer membrane protein (OMP) assembly. The C‐ and N‐termini of BamA fold into trans‐membrane β‐barrel and five soluble POTRA domains respectively. Detailed characterization of BamA POTRA 1 missense and deletion mutants revealed two competing OMP assembly pathways, one of which is followed by the archetypal trimeric β‐barrel OMPs, OmpF and LamB, and is dependent on POTRA 1. Interestingly, our data suggest that BamA also requires its POTRA 1 domain for proper assembly. The second pathway is independent of POTRA 1 and is exemplified by TolC. Site‐specific cross‐linking analysis revealed that the POTRA 1 domain of BamA interacts with SurA, a periplasmic chaperone required for the assembly of OmpF and LamB, but not that of TolC and BamA. The data suggest that SurA and BamA POTRA 1 domain function in concert to assist folding and assembly of most β‐barrel OMPs except for TolC, which folds into a unique soluble α‐helical barrel and an OM‐anchored β‐barrel. The two assembly pathways finally merge at some step beyond POTRA 1 but presumably before membrane insertion, which is thought to be catalysed by the trans‐membrane β‐barrel domain of BamA.     

Characterisation of YtfM, a second member of the Omp85 family in Escherichia coli

Omp85 proteins form a ubiquitous protein family, members of which are found in all Gram-negative bacteria. Omp85 of Neisseria meningitidis and YaeT of Escherichia coli are shown to be essential for outer membrane biogenesis. Interestingly, there exists a homologue to YaeT in E. coli and many proteobacteria, denoted YtfM, the function of which has not been described yet. Like YaeT, YtfM is predicted to consist of an amino-terminal periplasmic domain and a membrane-located carboxy-terminal domain. In this study, we present a first characterisation of YtfM by comparison to YaeT concerning structural, biochemical and electrophysiological properties. Furthermore, a knockout strain revealed that ytfM is a non-essential gene and lack of the protein had no effect on outer membrane composition and integrity. The only observable phenotype was strongly reduced growth, indicating an important role of YtfM in vivo.     

The Omp85 protein of Neisseria meningitidis is required for lipid export to the outer membrane

In Gram-negative bacteria, lipopolysaccharide and phospholipid biosynthesis takes place at the inner membrane. How the completed lipid molecules are subsequently transported to the outer membrane remains unknown. Omp85 of Neisseria meningitidis is representative for a family of outer membrane proteins conserved among Gram-negative bacteria. We first demonstrated that the omp85 gene is co-transcribed with genes involved in lipid biosynthesis, suggesting an involvement in lipid assembly. A meningococcal strain was constructed in which Omp85 expression could be switched on or off through a tac promoter-controlled omp85 gene. We demonstrated that the presence of Omp85 is essential for viability. Depletion of Omp85 leads to accumulation of electron-dense amorphous material and vesicular structures in the periplasm. We demonstrated, by fractionation of inner and outer membranes, that lipopolysaccharide and phospholipids mostly disappeared from the outer membrane and instead accumulated in the inner membrane, upon depletion of Omp85. Omp85 depletion did not affect localization of integral outer membrane proteins PorA and Opa. These results provide compelling evidence for a role for Omp85 in lipid transport to the outer membrane.