Omp85 from the thermophilic cyanobacterium Thermosynechococcus elongatus differs from proteobacterial Omp85 in structure and domain composition

Abstract

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 A 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.

FIGURE 1.

Domain composition of POTRA domain proteins investigated in this study (not to scale). POTRA domains are represented by open circles and are numbered starting from the N terminus. FtsQ and DivIB include an N-terminal transmembrane helix, which anchors the protein in the cytoplasmic membrane. All other proteins are outer membrane proteins with a β-barrel domain illustrated by a dark gray box. *, TPSS of T. erythraeum is fused to its substrate and has a N-terminal hemagglutinin-like domain. §, most Omp-Patatins include only one POTRA domain. #, most cyanobacterial Omp85s have a proline-rich (10–40%) region of variable length and N-terminal to the first POTRA domain.

FIGURE 2.

A, cluster map of the POTRA domains of Sam50, Toc75-III, and Toc75-V, cyanobacterial Omp85 (cyOmp85), proteobacterial Omp85, cyanobacterial (cyTPSS), and proteobacterial TPSS proteins. Each dot represents a single sequence. The intensity of the connecting lines is proportional to the quality of the pairwise BLAST p-value ranging from light gray (1 × 10⁻²) to black (1 × 10⁻²⁰). The POTRA domains cluster according to their position in related proteins. The N-terminal POTRA domains of cyOmp85 (1 of 3; 1/3), Toc75 (1/3) and Omp85 (1/5) cluster close together. Likewise, the C-terminal POTRA domains (3/3 or 5/5, respectively) of these proteins, together with the single POTRA domain of Sam50, cluster close together. Note, however, that there is no direct connection between the chloroplast and cyanobacterial clusters on the one hand, and the mitochondrial clusters on the other hand, illustrating the huge evolutionary distance between them. Sam50 sequences disperse in a number of subclusters that represent the different kingdoms of eukaryotic life. The central POTRA domains of Omp85 (2/5, 3/5, and 4/5) and cyOmp85 (2/3) cluster further away; for some POTRA domains, the sequences from the α-Proteobacteria form discrete subclusters marked with α. The N-terminal TPSS and cyTPSS POTRAs (1/2) clusters in close proximity to the central POTRA (2/3) of cyOmp85. The C-terminal POTRA domain of cyTPSS and proteobacterial TPSS cluster apart from each other, indicating sequence divergence, most likely because of optimization on the specific substrates. B, cluster map of the β-barrel domains of Sam50, Toc75-III, and -V, cyanobacterial Omp85 (cyOmp85), Fusobacterium sp. Omp85 (fuOmp85), Omp85, cyanobacterial (cyTPSS), and proteobacterial TPSS proteins. Each dot represents a single sequence. The intensity of the connecting lines is proportional to the quality of the pairwise BLAST p-value ranging from light gray 1 × 10⁻⁴ to black (1 × 10⁻⁴⁰). The sequences cluster in functional groups, with Sam50, Omp85, and TPSS showing satellite subclusters. Toc75-III, the translocation channel in the chloroplast outer envelope, forms a separate cluster apart from Toc75-V and cyOmp85. Together, these clusters do not directly connect to the mitochondrial (Sam50) clusters, comparable with what is seen for the POTRA domains, above. Proteobacterial TPSS are almost exclusively linked with cyTPSS. The β-barrels of Fusobacterium sp. and the Clostridiales Veillonella parvula and Selenomonas flueggi cluster halfway between cyOmp85 and Omp85, due to possible recombination after horizontal gene transfer. Overall, the p-values indicate a high degree of homology between the mitochondrial Sam50 and Omp85 as well as chloroplast Toc75, cyOmp85, fuOmp85, and Omp85 β-barrels, providing strong support for the endosymbiont theory.

FIGURE 3.

Electrophysiological characterization of TeOmp85 and TeOmp85-C (which represents only the β-barrel domain of TeOmp85). A, current traces of TeOmp85 showing the low conductance state (upper panel) and the high conductance state (lower panel), at 100 mV. B, current traces of TeOmp85-C showing the low conductance state (upper panel) and the high conductance state (lower panel) at 50 mV. C, I/V plots of TeOmp85, showing the low (upper left), medium (upper right), and high (lower left) conductance state. Lower right, all three conductance states. D, TeOmp85-C, multiple channels inserting into the membrane. Stepwise increase of the current flow, usually leading to membrane rupture within minutes. E, conductance histogram of TeOmp85, measured at 100 mV, 1559 events. F, conductance histogram of TeOmp85-C, measured at 50 mV, 936 events.

FIGURE 4.

Structure of the N-terminal domain of TeOmp85. A, two ribbon plots of the TeOmp85 domain architecture rotated by 180° show the three POTRA domains PD1, PD2, and PD3. The structure is color-coded from the N terminus (NT, blue) to the C terminus (CT, red). Interfaces connecting the POTRA domains are enlarged, and residues contributing to the interface contacts are labeled. B, schematic view of the POTRA domain fold. A β-sheet consisting of three β-strands is flanked by two α-helices, yielding a topology of β1-α1-α2-β2-β3. C, structure superposition of the three TeOmp85 POTRA domains shown in blue (PD1), green (PD2), and red (PD3). An unusual protrusion present only in the β2 strand of PD2 is marked by an arrow. D, superposition of the two N-terminal POTRA domains of TeOmp85 (TeOmp85-PD1) and E. coli Omp85 (BamA, EcOmp85-PD1). Identical residues identified in the superposition are highlighted in stick representation. E, structure alignment of the entire FhaC Omp85-like export protein (in blue) with the N-terminal portion of TeOmp85 (in orange). POTRA domains one and two of the integral transporter align well with POTRA domains PD2 and PD3 of the TeOmp85 protein. The potential substrate-binding site located in the first POTRA domain of FhaC is marked. The overall representation is a good model for the full cyOmp85 structure.

FIGURE 5.

Analysis of conserved residues in the N-terminal domain and model of the C-terminal domain. A, surface representation of the N-terminal domain with two views of TeOmp85 rotated by 180°. Conserved residues are marked in blue on the surface. Patches of high conservation are mostly found at the C-terminal end of the structure, which presumably is involved in the interactions with the β-barrel part of the protein. Another region of high conservation is seen at the interface between PD2 and PD3. Individual conserved residues at this interface are marked in orange (for PD2) and blue (for PD3). B, surface representation of the C-terminal domain model of TeOmp85 (based on the structure of FhaC) in two different views. Note two areas of high conservation. One area is exposed in the loop between strands β2 and β3. Residues comprising this area are presumably involved in interactions with the C-terminal POTRA domain. Close by, a second region of high conservation is formed by the first β-strand together with the terminal three β-strands. As for most β-barrel proteins of the bacterial outer membrane, the C terminus is highly conserved (marked GIGERF) and carries a phenylalanine residue (marked F) similar to bacterial porins. C, representation of the surface exposed to the interior of the channel. The highly conserved loop L6 is shown aligned to another conserved patch (CONS1) at the inner surface of the barrel.