1.B.33 The Outer Membrane Protein Insertion Porin (Bam Complex) (OmpIP) Family
Gram-negative bacterial outer membrane proteins (OMPs) are assembled from the periplasm into the outer membrane in a process that is usually, but not always, dependent on the BAM or OmpIP complex (Dunstan et al. 2015). A large outer membrane antigen in Neisserial species, Omp85 (TC #1.B.33.1.1, 797 aas) (Genevrois et al., 2003) is homologous to the protective surface antigen D15 precursor in Haemophilus influenzae (TC #1.B.33.1.2; 797 aas). These bacterial proteins are distantly related to the chloroplast import-associated β-barrel channel, IAP75 (TC #1.B.33.2.1; 809 aas), a constituent of the chloroplast envelope protein translocase (CEPT or Tic-Toc) family (TC #3.A.9). IAP75 is a β-barrel porin in the outer membrane of pea chloroplasts (Ertel et al., 2005). Omp85 is also distantly related to the yeast mitochondrial Sorting and Assembly Machinery (SAM) constituent, SAM50 (Kozjak et al., 2003). The SAM complex in yeast mitochondria consists of at least three proteins, SAM50, SAM35 and MAS37 (Kozjak et al., 2003; Milenkovic et al., 2004; Wiedemann et al., 2003). It is required for the assembly of outer membrane β-barrel proteins in mitochondria (Tommassen, 2010). Omp85 homologues consist of two domains: an N-terminal periplasmic domain with POTRA repeats, and the C-terminal β-barrel domain in the outer membrane. Omp85 proteins are distantly related to core proteins of 2-partner secretion systems as well as plastid Toc75 proteins (Gentle et al., 2005). The BAM complex may function in conjunction with the Translocation and assembly module 1 (TamAB) for the insertion and folding of certain proteins such as autotransporters (Heinz et al. 2015). The mechanism of protein insertion via the Bam system has been reviewed (Konovalova et al. 2017).
The functionally characterized homologue in Neisseria meningitidis, Omp85, is essential for bacterial viability. Unassembled forms of various outer membrane proteins accumulate when Omp85 is depleted (Voulhoux et al., 2003). Moreover, immunofluorescence microscopy showed decreased surface exposure of outer membrane proteins, particularly at the cell division planes. Homologues of Omp85 are present in all Gram-negative bacteria examined (Voulhoux et al., 2003). Signals in bacterial β-barrel proteins are reported to be functional in eukaryotic cells for targeting to and assembly in mitochondria (Walther et al., 2009). Bacterial Omp85 proteins are characterized by a periplasmic domain containing five repeats of polypeptide transport-associated (POTRA) motifs. Gatzeva-Topalova et al., 2008 reported the crystal structure of a periplasmic fragment of YaeT (the E. coli Omp85) containing the first four POTRA domains in an extended conformation. Analyses of the YaeT structure revealed conformational flexibility around a hinge point between the POTRA2 and 3 domains. YaeT (BamA), the central BAM subunit composed of a transmembrane beta-barrel domain linked to periplasmic PORTRA domains, is thought to bind nascent OMPs and undergo conformational cycling to catalyze OMP folding and insertion. Conformational cycling of hinge motions between POTRA2 and POTRA3 is required for biological function (Warner et al. 2017).
The cell envelope of the thermophilic bacterium Thermus thermophilus HB27 is multilayered including an outer membrane that is not well characterized. Neither the lipid composition nor the integral membrane proteins are characterized. The genome encodes one Omp85-like protein, TtOmp85, representing an ancestral type of this family. Nesper et al (2008) overexpressed TtOmp85 in T. thermophilus and purified it from the native outer membranes. In the presence of detergent, purified TtOmp85 existed mainly as a monomer composed of two stable protease resistant modules. Circular dichroism spectroscopy indicated beta-sheet secondary structure. Electron microscopy of negatively stained lipid embedded TtOmp85 revealed ring-like structures with a central cavity of approximately 1.5 nm in diameter. Single channel conductance recordings indicated that TtOmp85 forms ion channels with two different conducting states, characterized by conductances of approximately 0.4 nS and approximately 0.65 nS, respectively (Nesper et al., 2008).
Generally in Gram-negative bacteria, lipopolysaccharide (LPS) and phospholipids (PLs) destined for the outer membrane are made in the inner membrane. Genevrois et al. (2003) have reported that the Omp85 structural gene is cotranscribed with genes involved in lipid biosynthesis. Depletion of Omp85 results in accumulation of LPS and PL in the inner membrane and loss from the outer membrane. The effects on lipids were reported to precede the effects on outer membrane protein (PorA and Opa) insertion, suggesting that the latter effects were secondary to the effects on LPS and PL translocation (Genevrois et al., 2003). However, Doerrler and Raetz (2005) came to the opposite conclusion when studying the effect of yaeT mutants in E. coli. They concluded that YaeT functions primarily in protein insertion into the outer membrane. A different protein, OstA or Imp (784 aas in E. coli; TC #1.B.42; P31554) may mediate lipopolysaccharide export (Bos et al., 2004).
Normally OMPs are translocated into the periplasm via the Sec translocase (TC #3.A.5). They are believed to fold in the periplasm before being inserted into the outer membrane. Folding is stimulated by the small (82 aa) periplasmic chaperone protein SurA (P21202), a peptidyl prolyl cis-trans isomerase (PPIase) also called Rotamase C or parvulin (POA9L5), by lipopolysaccharide, which normally comprises the outer leaflet of the outer membrane, and by two other periplasmic/outer membrane proteins, Skp (OmpH; HlpA; P11457; 161 aas) and another PPIase, FkpA (P45523; 270 aas) (Missiakas et al., 1996). Still other proteins may be involved. It is even possible that an energy source will prove to be required.
In E. coli, a multiprotein complex has been shown to be required for outer membrane biogenesis (Wu et al., 2005). This complex includes the Omp85 homologue, YaeT, a lipoprotein, YfgL, and three other proteins, YfiO, NlpB and SmpA (Kim et al., 2007; Sklar et al., 2007).These proteins function in outer membrane protein assembly. Components YaeT, YfiO and YfgL interact directly, and NlpB interacts with YfiO. The lipoprotein, YfiO, stabilized the complex, is essential for complex formation and is required for β-barrel insertion (Malinverni et al., 2006). YfgL and YfiO make direct but independent physical contacts with YaeT. Whereas the YaeT-YfiO interaction needs NlpB and SmpA for complex stabilization, the YaeT-YfgL interaction does not. The periplasmic chaperone, SurA, binds to YaeT (or another complex member) without going through YfgL (Vuong et al., 2008). YaeT serves as a receptor for short tailed Shiga-toxin-encoding (Stx) bacteriophage (Smith et al., 2007). The reconstituted E. coli Bam complex catalyzes multiple rounds of beta-barrel assembly (Hagan and Kahne, 2011).
The protein complex formed of YfgL together with NlpB, YfiO, SmpA and YaeT insert proteins into the outer membrane. Without YfgL, the levels of OmpA, OmpF, and LamB are significantly reduced. When cells are depleted of YaeT or YfiO, levels of all outer membrane proteins examined are severely reduced. Thus, all assembly pathways use the YaeT/YfiO complex (Charlson et al., 2006). Outer membrane protein assembly has been reconstituted from purified components (Hagan et al., 2010). Assembly occurs without an energy source, but requires a soluble chaperone in addition to the multiprotein assembly complex.
YaeT of Escherichia coli, has one or more polypeptide transport-associated (POTRA) domains. The crystal structure of a periplasmic fragment of YaeT (Kim et al., 2007) revealed the POTRA domain fold and suggested a model for how POTRA domains can bind different peptide sequences, as required for a machine that handles numerous β-barrel protein precursors. Analysis of POTRA domain deletions showed which are essential and provided a view of the spatial organization of this assembly machine (Kim et al., 2007). Three dimensional structural analyses reveal how multiple POTRA domains may recruit substrates from the periplasm and help insert them into the outer membrane (Knowles et al., 2008). The folding and membrane insertion of multimeric OMPs has been reviewed (Leo et al. 2015).
The pore-forming regions of Omp85 proteins are restricted to the C-termini. Based on phylogenetic analyses, the pore-forming domains display different evolutionary relationships than the N-terminal domains. The N-terminal domains are involved in gating of the pore, recognize the substrate proteins, participate in complex formation, and take part in homo-oligomerization. The differences in the phylogenies of the two domains are explained by different functional constraints acting on the two regions. The pore-forming domain is divided into two functional regions where the distal C terminus forms a dimeric pore. Based on functional and phylogenetic analyses, an evolutionary scenario that explains the origin of the contemporary translocon has been proposed (Bredemeier et al., 2007).
Arnold et al. (2010) studied the Omp85 from the thermophilic cyanobacterium Thermosynechococcus elongatus and found that its Omp85 is more closely related to the chloroplast homologue Toc75 than to proteobacterial Omp85. They solved the structure of the periplasmic part of the protein to 1.97 A resolution, and demonstrated that in contrast to Omp85 from E. coli, the protein has only three, not five, polypeptide transport associated (POTRA) domains (P1, P2, and P3), which recognize substrates and generally interact with other proteins in larger complexes. They modeled how these POTRA domains are attached to the outer membrane. A hinge is present between P1 and P2, but P2 and P3 are fixed in orientation. Koenig et al., (2010) defined interfaces for protein-protein interaction in P1 and P2. P3 possesses an extended loop unique to cyanobacteria and plantae, which influences pore properties.
A gene encoding the outer membrane lipoprotein, OmlA, also called SmpA, a constiutent for the BAM complex, from the bacterial phytopathogen Xanthomonas campestris pv. phaseoli when knocked out showed increased susceptibility to novobiocin and coumermycin, antibiotics with gyrase inhibitory activity. The omlA mutant accumulated novobiocin and was more sensitive chloramphenicol, SDS, and menadione (a superoxide generator). The susceptibility of the mutant to unrelated chemicals indicated a general role for OmlA in maintaining membrane integrity. Transcription of omlA was downregulated in the presence of both gyrase inhibitors, suggesting that DNA supercoiling might regulate the synthesis of OmlA (Fuangthong et al. 2008). The 3-d structure of OmlA has been solved by NMR (Vanini et al. 2006).
As noted above, the β-barrel assembly machinery (BAM) complex is responsible for the biogenesis of β-barrel membrane proteins with homologous complexes found in mitochondria and chloroplasts. Noinaj et al. 2013 described the structure of BamA, the central, essential component of the BAM complex, from two species of bacteria: Neisseria gonorrhoeae and Haemophilus ducreyi. BamA consists of a large periplasmic domain attached to a 16-strand transmembrane β-barrel domain. Three structural features shed light on the mechanism by which BamA catalyzes β-barrel assembly. First, the interior cavity is accessible in one BamA structure and conformationally closed in the other. Second, an exterior rim of the β-barrel has a distinctly narrowed hydrophobic surface, locally destabilizing the outer membrane. Third, the β-barrel can undergo lateral opening, suggesting a route from the interior cavity in BamA into the outer membrane (Noinaj et al. 2013).
An eight-stranded outer membrane beta-barrel protein, TtoA (TC#1.B.6.8.1), is inserted and folded into liposomes by the Omp85 homologue of Thermus thermophilus (Estrada Mallarino et al. 2015). The channel conductance of this Omp85 protein was measured in black lipid membranes, alone and in the presence of peptides comprising the sequence of the two N-terminal and the two C-terminal beta-strands of TtoA. Only with the latter could a long-lived channel that exhibits conductance levels higher than those of the Omp85 protein alone be observed. Thus, unfolded outer membrane protein, after docking with its C-terminus, penetrates into the transmembrane beta-barrel of the Omp85 protein and augments its beta-sheet at the first strand. Augmentation with successive beta-strands leads to a compound, dilated barrel of both proteins (Estrada Mallarino et al. 2015).
Omp85 transporters and Two Partner Secretion (TPS) systems have the same conserved architecture, with POTRA domains that interact with substrate proteins, a 16-stranded transmembrane beta barrel, and an extracellular loop, L6, folded back in the barrel pore. Guérin et al. 2015 showed that the L6 loop of FhaC changes conformation and modulates channel opening. Those conformational changes involve breaking the conserved interaction between the tip of L6 and the inner beta-barrel wall. The membrane-proximal POTRA domain also exchanges between several conformations, and the binding of FHA displaces this equilibrium. There is dynamic, physical communication between the POTRA domains and L6 within the beta barrel (Guérin et al. 2015).
The structure of the 200-kilodalton five-component BAM complex from E. coli has been solved, revealing that binding of BamCDE modulates the conformation of BamA, the central component. This interaction may serve to regulate the activity of the BAM complex. The periplasmic domain of BamA was crystalized in a closed state that prevents access to the barrel lumen. This might indicate that substrate OMPs may not be threaded through the barrel during biogenesis. Further, conformational shifts in the barrel domain lead to opening of the exit pore and rearrangement at the lateral gate (Bakelar et al. 2016).
Homologous Omp85 proteins are essential for membrane insertion of β-barrel precursors. Precursors are apparently threaded through the Omp85-channel interior and exit laterally. Höhr et al. 2018 mapped the interaction of a precursor in transit with the mitochondrial Omp85-channel Sam50 in the native membrane environment. The precursor is translocated into the channel interior, interacts with an internal loop, and inserts into the lateral gate by β-signal exchange. Transport through the Omp85-channel interior followed by release through the lateral gate into the lipid phase represents a basic mechanism for membrane insertion of β-barrel proteins (Höhr et al. 2018).
In mitochondria, β-barrel outer membrane proteins mediate protein import, metabolite transport, lipid transport, and biogenesis. The Sorting and Assembly Machinery (SAM) complex consists of three proteins that assemble as a 1:1:1 complex to fold β-barrel proteins and insert them into the mitochondrial outer membrane. Diederichs et al. 2020 reported cryoEM structures of the SAM complex from Myceliophthora thermophila, which show that Sam50 forms a 16-stranded transmembrane beta-barrel with a single polypeptide-transport-associated (POTRA) domain extending into the intermembrane space. Sam35 and Sam37 are located on the cytosolic side of the outer membrane, with Sam35 capping Sam50, and Sam37 interacting extensively with Sam35. Sam35 and Sam37 each adopt a GST-like fold, with no functional, structural, or sequence similarity to their bacterial counterparts. Structural analysis shows how the Sam50 beta-barrel opens a lateral gate to accommodate its substrates (Diederichs et al. 2020).
The transport reaction catalyzed by the OmpIP (BAM) complex is:
OMPs (periplasm) → OMPs (outer membrane)