Structure model of BamA from E. coli and BamA complex formation. A, the model of full-length BamA from E. coli is shown inserted in a schematically depicted OM. The model was prepared on the basis of the FhaC transporter structure (PDB code 2QDZ) comprising a β-barrel domain and two N-terminal POTRA domains (18). The membrane spanning the β-barrel part of BamA was modeled using FhaC coordinates and the Modeler program (63). Further positioning of BamA POTRA domains (P4 and P5, PDB code 3OG5) was accomplished by the superposition onto the FhaC POTRA domains (33, 56). This initial model was then extended using the BamA crystal structure comprising the POTRA P1-P4 domains (PDB code 3EFC) by a structure alignment of the overlapping P4 domains (56). The full-length model of BamA (orange) was subsequently used to localize conserved residues that were marked in blue to detect a possible accumulation of residues (e.g. (1) in strands β16, β17, and β18). This part of the structure is highlighted by a close-up view, with the two proline residues emphasized by red dots. Additional parts of the protein are conserved: (2), the junction between POTRA and the barrel domain and (3), the longest loop extending into the barrel domain. B, SDS-PAGE analysis demonstrating the complex formation of BamA (A), BamB (B), and BamD (D). In lane 1, BamB and BamD were applied to Ni-NTA chromatography. Lane 2 shows the elution of a mixture containing BamA, BamB, and BamD, with BamB comprising the His tag. In lane 3, the mixture of BamA and BamD was applied, with BamA as the His-tagged protein. Lane 4 shows a mixture of partially unfolded but soluble and His-tagged BamA in octyl-polyoxyethylene detergent incubated with BamB. A mixture of folded BamA and BamB was applied after Ni-NTA separation. Proteins comprising the His tag are marked in red.

Structure of BamB and comparison with related structures and analysis of conservation. A, structure representation of BamB in side view perspective with the putative membrane-anchoring lipid connected by a flexible linker (dashed lines) to the lipoprotein. The structure is shown in schematic representation, with the N and C terminus (NT/CT) and important loops (L1, L2) labeled. The area interacting with BamA is indicated together with the top and bottom face assignment. Structures in the top panel on the right display the eight WD40 repeats (B1-B8) together with residues known to be essential for BamA interactions (20). These functional residues are almost all located on the top face of the protein and cluster along the central pore of the protein. In the surface representation of BamB, the localization of functional residues is indicated together with the secondary structure assignment of the first WD40 repeat (encircled numbers 1–4). B, superposition of BamB with the Skp-Fbw7-CyclinEdegN complex (PDB code 2OVR), which was cocrystallized with an N-terminal N-degron peptide (PEP, marked in dark blue with the residual C-α position indicated by yellow dots). The peptide is bound on top of the scaffold architecture provided by the extended loop structures (37). C, close-up of the peptide binding site in the FCE complex with structurally important residues emphasized. The localization of amino acids known to be important for function in BamB is assigned (Leu-173, Leu-175, Arg-76), all of which are located in close proximity to residues of the N-degron peptide (here only the residue types are marked with R and R' for arginine residues involved in peptide binding). Notably, the N-degron peptide and related cocrystal structures of WD40 propellers often display proline residues (here specifically marked with P and red dots).

Structure analysis and conservation patterns of BamB. A, analysis of conserved residues using the surface representation of BamB. In the upper panel, the protein surface is shown in top and bottom view (related by a rotation of the molecule around the x axis). Conserved residue patches on the surface are marked in light blue, and important positions facing the surface are marked with numbers. Residue types are color-coded with dots: green, hydrophobic residues; blue, charged residues. In the lower panel, the BamB surface in side view is shown from two different positions that are related by a rotation of the molecule by 180 degrees around the y axis. A second but smaller conserved patch on the outer rim surface appears for residues Gln-206-Met-209. B, size exclusion chromatography of BamB showing the size distribution of the protein as monomers and higher oligomers (1, 4, and 6). C, superposition of BamB structures from this work and recently published structures (38, 40, 54). The three structures show a small structural divergence of 0.6 and 0.5 r.m.s.d., respectively.

Structure of the BamC scaffold evolved through gene duplication. A, schematic assignment of the N- and C-terminal domains (BamC_ND and BamC_CD) to the BamC sequence. BamC_ND is color-coded orange, and BamC_ND is marked in red. These two domains were defined through limited proteolysis, which led to the cleavage of 14 residues in between these domains. B, two structure views of BamC_ND, both related by a rotation of 180 degrees around the y axis. The structure comprises a α/β-mixed fold arranged as an αβ-sandwich domain. The individual secondary structure elements and domain borders are marked (β1-β5 and α1 and α2). NT, N terminus; CT, C terminus). C, domain architecture of BamC_CD with two views related by the rotation of 180 degrees around the y axis. Secondary structure elements are marked as for the N-terminal domain in B. D, superposition of the N- and C-terminal domains. Although the sequence similarity of the domains is low, the structural conservation is clearly visible. The main difference in structure between both domains is the presence of an additional N-terminal β-strand in the BamC_CD domain. E, architecture of the full-length protein with the outer membrane delineated. The N-terminal residues of the full-length protein are predicted to be unstructured. The two-domain structure was assembled inside the ∼10-nm-long elongated protein envelope generated by SAXS data analysis. Among all four lipoproteins, BamC shows the longest unstructured N-terminal sequence, approximately 70 residues.

Structure of the oligomeric intertwined BamE complex. A, structure of the dimeric BamE protein unit in side and top view (tilted by 90 degrees around the x axis). Secondary structure elements and termini are marked accordingly. B, structure of the hexameric protein complex, which is formed by intercalation of three dimers (light blue and red). Two monomers (orange and blue) are indicated in surface representation to show the strong knot-like interface. C, representation of the hexameric protein complex from two different orientations, with a monomer represented as surface representation. Conserved residues are plotted onto the surface (cyan) to mark the interface between the two monomers. D, the same surface view as in C, and residues involved in interface stabilization between monomers are marked with different colors depending on the monomer-monomer interface contacts. Taking the conservation fingerprint together with the stabilizing residues of the hexameric complex indicates that the dimeric assembly is of importance, whereas the hexameric complex may assemble under different conditions.

Protein oligomerization states of BamE and interaction studies of BamD with OMP peptides. A and B, size exclusion chromatography runs of truncated (residues 21–94 of the full-length protein) and the full-length BamE (including the lipid anchor). Molecular masses indicate the oligomeric state (hexameric for the full-length protein and ∼tetrameric for the shortened version) of both species in solution (oligomerization number size is marked with 1, 4, and 6). C and D, further confirmation of the oligomeric state of BamE by glutaraldehyde-mediated cross-linking of truncated BamE (residues 21–94) (C) and full-length BamE (D). Protein samples are shown before (1) and after (2) cross-linking. Samples were subjected to SDS-PAGE and showed a ladder-like appearance, indicating the presence of oligomers (up to hexamers) in solution. E, formation of a BamD-peptide complex as shown by cross-linking experiments. Peptides used for this experiment were derived from the structure of the Hia-autotransporter, and glutaraldehyde has been used a cross-linker (12). The two 26-residue-long peptides comprising β-strands β1, β2, and β3, and β4 were used to be cross-linked with the truncated form of BamD (lanes 1-4) and the full-length version of the protein (lanes 5-8). Lanes 1 and 2 show the protein alone before and after cross-linking. Lanes 3 and 4 demonstrate cross-linking to the two different peptides. The same arrangement is shown in lanes 5 and 6. The full-length protein was cross-linked before the full-length protein was cross-linked to the individual peptides (lanes 7 and 8).

The essential BamD protein represents a TPR scaffold. A, structure representation of BamD shown from two different sides. Secondary structure elements of the exclusively α-helical protein are marked (α1-α10). The C-terminal extension of a second BamD protein is marked in stick representation. B, close-up of the TPR_1–3 binding site. The C-terminal peptide is marked in stick representation (blue), and interactions between the peptide and TPR scaffold are marked by dashed lines (important residues are numbered and given in stick presentation). C, the same orientation of the peptide as in B, but additional information of OMP_CT sequences is provided. The residues of the peptide carry the nomenclature of x (last residue) to x-5. Conservation of residues as the “OMP fingerprint” for E. coli OMP proteins is given to compare residues supposedly binding at the same site with the actual sequence of the C-terminal BamD portion. D, conservation analysis of BamD sequences. The protein visualized from two sides (identical to A) shows three conserved patches (I-III), residues are numbered and marked in blue), one of which comprises residues located in the TPR domain fold. The second patch is nearby and may be involved in SurA or BamA binding. The third patch is localized on the opposite side of the protein, with a significant number of tyrosine residues involved.

Model of the β-barrel assembly machinery. Proteins destined for the outer membrane are delivered to the BAM complex with the help of periplasmic chaperones. Most importantly, SurA has been shown to bind OMP peptides and to deliver substrates for processing by the BAM complex (6, 9). The entire BAM complex as a model is presented with the five compounds BamA-BamE color-coded (BamA, orange; BamB, blue; BamC, magenta; BamD, red; BamE, green). A possible scenario for OMP biogenesis on the basis of the data presented in this paper is the substrate delivery of a putative OMP (with the OMP_CT marked in yellow) from SurA to BamD that can recognize both the BamA protein and OMP peptides. The substrate is presumably transferred to the BAM complex and subsequently inserted into the outer membrane.