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The structure of the β-barrel assembly machinery complex

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Science  08 Jan 2016:
Vol. 351, Issue 6269, pp. 180-186
DOI: 10.1126/science.aad3460

Going in with a BAM

Integral membrane proteins in bacterial outer membranes play roles in nutrient import and infectivity. These proteins are folded into a barrel shape composed of β-strands and inserted into the outer membrane by the β-barrel assembly machinery (BAM) complex. Bakelar et al. determined the crystal structure of a four-component BAM subcomplex. The structure of a central β barrel in BAM changes in the presence of the accessory components to create a lateral opening that may be involved in how BAM inserts proteins into the outer membrane.

Science, this issue p. 180

Abstract

β-Barrel outer membrane proteins (OMPs) are found in the outer membranes of Gram-negative bacteria and are essential for nutrient import, signaling, and adhesion. A 200-kilodalton five-component complex called the β-barrel assembly machinery (BAM) complex has been implicated in the biogenesis of OMPs. We report the structure of the BAM complex from Escherichia coli, revealing that binding of BamCDE modulates the conformation of BamA, the central component, which may serve to regulate the BAM complex. The periplasmic domain of BamA was in a closed state that prevents access to the barrel lumen, which indicates 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.

Gram-negative bacteria contain both an inner membrane (IM) and an outer membrane (OM) that serve important roles in nutrient import, cell signaling, waste export, and protection. Integral membrane proteins in the IM all have an α-helical fold consisting of one or more α-helices. In the OM, however, integral membrane proteins have a β-barrel fold consisting of 8 to 26 antiparallel β-strands. In pathogenic strains of bacteria, some outer membrane proteins (OMPs) can also serve as virulence factors that mediate infection. OMPs are only found in the OMs of Gram-negative bacteria, mitochondria, and chloroplasts (1, 2).

The exact mechanism explaining how OMPs are folded and inserted into the OM remains unknown; however, studies have identified a general pathway and conserved machinery that is responsible for the biogenesis of OMPs (35). A majority of these advances have been made by working with Gram-negative bacteria (3, 6, 7). Here, the nascent OMPs are first synthesized in the cytoplasm with an N-terminal leader sequence that directs them to the Sec translocon for transport across the IM, into the periplasm (Fig. 1A). Chaperones, such as SurA and Skp, then further escort the nascent OMPs to a multicomponent complex called the β-barrel assembly machinery (BAM) complex, which is responsible for folding and inserting OMPs into the OM (4, 8). In Escherichia coli, the BAM complex consists of five components, BamA, B, C, D, and E. BamA, a 16-stranded OMP, is the central component of the complex and is conserved both in mitochondria and chloroplasts, whereas BamB to BamE are all lipoproteins. BamA and BamD are essential for viability; however, all components are required for efficient OMP folding and insertion (911). BamB and BamD interact directly with BamA via nonoverlapping binding sites, whereas BamC and BamE interact directly with BamD to stabilize the complex (9, 12).

Fig. 1 The structure of the BAM complex.

(A) The pathway for the biogenesis of β-barrel OMPs in Gram-negative bacteria. (B) A membrane view of the structure of the full BAM complex, formed from merging the crystal structures of BamACDE and BamAB (PDB ID 4PK1). The bottom panel shows the periplasmic view, rotated 90° along the x axis relative to the top panel. BamA is shown in green, BamB in gray, BamC in blue, BamD in gold, and BamE in purple. (C) The β-barrel domain of BamA undgoes a dramatic conformational change along strands β1 to β8, which can be seen here. This panel is rotated ~90° along the y axis relative to the top view of panel (B). The overall measurements of the BAM complex are ~115 by 115 by 115 Å.

Structures of all the Bam components have now been reported, including partial complexes of BamAB and BamCD (7, 1323). The full-length structure of BamA from Neisseria gonorrhoeae revealed a large periplasmic domain consisting of five polypeptide transport–associated (POTRA) domains and a C-terminal 16-stranded β-barrel domain. Subsequent studies showed that the barrel domain of BamA opens laterally in the membrane, possibly to allow insertion of the substrate OMPs into the OM (16, 18, 24). BamB may serve as a scaffold to assist in the handoff of nascent OMPs from SurA to BamA, whereas BamC, BamD, and BamE may serve to regulate the function of BamA (14, 17, 25). The structures have offered clues to how each component may function within the complex; however, the lack of structural information regarding the fully assembled complex has hindered progress toward exploring the mechanism further. We used x-ray crystallography to solve the structure of the BamACDE subcomplex to 3.4 Å resolution and used the previously reported partial structure of BamAB to form a model of the fully assembled BAM complex from E. coli. The periplasmic domain of BamA was found in a closed state, which prevented access to the barrel lumen from the periplasm. Binding of BamCDE to BamA causes conformational twisting of strands β1 to β8 and leads to opening of the exit pore and structural rearrangement of the lateral opening site. These structural changes suggest that the role of BamCDE may be to modulate the conformational states of BamA, which regulate the BAM complex.

The BAM complex from E. coli was expressed from a single plasmid and purified as previously described with some modifications (supplementary methods) (11). SDS–polyacrylamide gel electrophoresis (SDS-PAGE) analysis verified the presence of the full complex, which produced a monodisperse peak as seen with size-exclusion chromatography (fig. S1). We crystallized the complex in C8E4 and collected data at the SER-CAT ID22 beamline at the Advanced Photon Source. The crystal structure of the complex was then solved by molecular replacement (fig. S2 and table S1). On the basis of crystal-packing analysis, it was clear that BamB was absent rather than being disordered. This was possibly due to proteolytic degradation during incubation. This was verified by SDS-PAGE analysis of crystals and of the original protein sample, both of which lacked BamB after extended incubation or storage (fig. S1). The crystal structure contains full-length BamA, BamD, and BamE but only the N-terminal flexible domain and the first globular helix-grip domain of BamC, with the second helix-grip domain presumably being disordered. To model the fully assembled BAM complex, we used the previously reported structure of the partial BamAB complex [Protein DataBank (PDB) ID 4PK1] (14) to dock BamB into our crystal structure by aligning along POTRA3 of BamA (Fig. 1, B and C; movie S1; and model S1). For all accessory lipoproteins, the N-terminal residues are positioned in close proximity to where the OM would sit; however, no lipid anchors were observable in our crystal structure.

BamCD in our structure aligned well with the previously reported complex (PDB ID 3TGO), which has a root mean square deviation (RMSD) of 1.25 Å across both chains (Fig. 2, A and B). BamE interacts with the opposite side of BamD [buried surface area (BSA) ~800 Å2] along the C-terminal end (Fig. 2A and tables S2 and S3). Although BamC and BamE interact with BamD via nonoverlapping binding sites, they make minimal contact with one another (BSA ~140 Å2). Residues 215 to 344 of BamC, consisting of a linker and the second helix-grip domain, were disordered in our crystal structure. Previous studies have shown that the two helix-grip domains of BamC are found on the outside of the cell (26), which suggests that one or both may interact with the surface-exposed loops of BamA. No interaction was observed in our structure, which indicates that if the two helix-grip domains do indeed interact with BamA, a membrane bilayer and/or substrate may be required to release BamC from BamD so it can be presented on the surface.

Fig. 2 Interactions of BamC and BamE with BamD.

(A) The complex structure of BamCDE is shown here with BamD in gold, BamC in blue, and BamE in purple. The TPR domains of BamD are indicated. The bottom panel is rotated 90° along the x axis relative to the top. (B) The interactions between BamC and BamD are nearly the same as that observed in the previously reported crystal structure (PDB ID 3TGO) with an RMSD of 1.25 Å across both chains. The bottom panel is rotated 90° along the x axis relative to the top. (C) View showing the interactions of BamE with BamD TPR4 and 5 through an extensive interface with a buried surface area of ~800 Å2, containing a mix of hydrogen bonds, salt bridges, and hydrophobic interactions. (D) A view from the top that is rotated 90° along the x axis to further illustrate the extensive binding interface.

The structure of BamD consists of five tetratricopeptide-repeat (TPR) motifs (15, 21, 27) and sits parallel to the membrane, with TPR4 and 5 forming the binding site for BamE, which is oriented perpendicular to the membrane (Fig. 2A). The binding interface agrees well with previously published work, where BamD was found to bind BamE along the interface containing residues R29, I32, F68, N71, T72, R78, and T92 (23, 28). The extensive binding interface has numerous hydrogen bonds using both side-chain and backbone atoms, as well as a salt bridge between D66 of BamE and K233 of BamD (Fig. 2, C and D). The binding is further strengthened by interactions of M64 and F68 of BamE with hydrophobic pockets in BamD. The interaction between BamC and BamE is mediated primarily by hydrophobic interactions; the loop of BamC helps to form the large hydrophobic pocket where F68 of BamE binds BamD (table S4).

In agreement with previous studies (29), BamD binds BamA along POTRA5 almost exclusively through TPR3 and 4 (BSA ~1100 Å2) (Fig. 3, A and B, and table S5). The extensive binding interface between BamD and POTRA5 of BamA is mediated by hydrogen bonds that use side-chain and backbone atoms and three salt bridges between residues H139, R197, and R188 of BamD and residues D358, E373, and D481 of BamA, respectively (Fig. 3, B and C). The salt bridge formed by R197 of BamD and E373 of BamA is central to this interaction and agrees with previous studies that implicate these residues in binding (30, 31). The extended loop of BamD TPR3 (residues 123 to 130) interacts with periplasmic loop 1 of BamA (residues 449 to 452; BSA ~120 Å2) (fig. S3). TPR1 and 2 of BamD also interact minimally with POTRA2 of BamA (BSA ~115 Å2) (Fig. 3, B and D), which suggests that BamD may also participate in modulating the conformation of the POTRA domains of BamA during OMP biogenesis.

Fig. 3 Interactions of BamD and BamE with BamA.

(A) BamD interacts primarily along POTRA5 of BamA via TPR4 and 5, but also along POTRA2 via TPR1 and 2, whereas BamE interacts along POTRA5. (B) A rotated view without the β-barrel domain of BamA highlighting the interacting regions. (C) View of the interaction of BamD with POTRA5 of BamA, showing an extensive binding interface with a buried surface area of ~1100 Å2, containing a mix of hydrogen bonds, salt bridges, and hydrophobic interactions. Residue R197 of BamD clearly forms a salt bridge with E373 of BamA. Residue D481 (green box) is from periplasmic loop 2 of BamA. (D) View of the interactions between TPR1 and 2 of BamD with POTRA2 of BamA. Although these interactions are minimal here, they could assist in modulating the conformation of BamA. (E) View of the interactions of BamE with POTRA5 of BamA, an extensive interaction with a buried surface area of ~750 Å2, containing a mix of hydrogen bonds, salt bridges, and hydrophobic interactions. (F) The N-terminal region of BamE was found anchored via hydrogen bonding interactions of residue Y28 to residues E521 and P518 of BamA periplasmic loop 3.

BamA interacts with BamD, and this is enhanced by the presence of BamE. However, no studies have shown that BamA also interacts with BamE (9, 12). In our crystal structure though, we observe not only the interaction of BamE with BamD but also an extensive interaction of BamE with POTRA5 of BamA (BSA ~750 Å2) (Fig. 3, B and E, and table S6). This binding interface is composed of numerous hydrogen bonds between side-chain and backbone atoms, as well as hydrophobic interactions primarily from W376 of BamA (BSA ~130 Å2). Residue Y28 of BamE interacts with periplasmic loop 3 of BamA, forming hydrogen bonds with P518 and E521, which anchor the N terminus of BamE in close proximity to the barrel domain of BamA. This may serve to orient BamCDE optimally for interacting with BamA (Fig. 3, A and F). The observation that BamE bridges BamD and BamA agrees well with previous work indicating that BamE stabilizes the interaction of BamD with BamA (12). Previous studies identified BamE residues involved in BamD binding (23); however, not all of these residues mapped to that interface in our structure. Instead, residues Y37, L38, T61, and L63 lie along the interface with POTRA5 of BamA, which suggests that binding of BamD may lead to conformational changes in BamE that promote association with BamA. Also, the BamE residues identified to interact with phosphatidylglycerol (PG) (G60, T70, N71, V76, and F95) are located within the periplasm as far as 35 Å from the OM (23). Therefore, it is unlikely that the PG-binding role of BamE contributes directly to the role of the BAM complex; however, it is still possible that the recruitment of PG in proximity to the BAM complex increases the efficiency of OMP biogenesis.

No structure of full-length BamA from E. coli has been reported previously, although a structure containing the β-barrel domain and POTRA5 is available (16). In this structure, POTRA5 was oriented away from the barrel domain in an open conformation allowing access to the barrel lumen from the periplasm (fig. S4). However, in our structure, POTRA5 is in a closed conformation relative to the β-barrel domain, fully occluding access to the barrel lumen from the periplasm. Compared with the BamA structure from N. gonorrhoeae (18), the POTRA domains rotate ~90° along the plane of the membrane upon interaction with BamCDE. POTRA5 makes numerous contacts with periplasmic loops 1, 2, 3, 4, and 6, with the most extensive interactions through periplasmic loops 1, 2, and 4 (fig. S5). This conformation constitutes a ~45° hingelike conformational change of POTRA5 to the closed state (Fig. 4A). POTRA5 interacts most extensively with periplasmic loop 4 (fig. S5 and movie S2), which is stabilized by a salt bridge between E396 (POTRA5) and R583 (periplasmic loop 4) and pi stacking between R421 (POTRA5) and Y585 (periplasmic loop 4). Conformational flexibility of the POTRA domains has been well documented (29, 32, 33). Therefore, it remains to be determined whether the association of BamCDE is solely responsible for the closed conformation.

Fig. 4 Conformational changes in the barrel domain of BamA.

(A) Superposition of BamA from our structure (green) with PDB ID 4C4V (cyan) reveals ~45° hingelike conformational change of the POTRA domains to a closed, periplasm occluded state. (B) Binding of BamCDE to BamA leads to an unprecedented twist of strands β1 to β8, with the most dramatic change emanating from strand β1 (~45°) and gradually diminishing until strand β9 (movie S2). This leads to opening of the exit pore (~15 Å by ~27 Å) and lateral gate. (C) In response to the shift of the barrel strands, an opening of surface loops 1, 2, and 3 was observed; however, surface loops 4, 5, 6, 7, and 8 were mostly unchanged. (D) Rotated view highlighting the strand shift along strands β1 to β8 of the barrel domain of BamA. (E) View of the strand shift along strands β1 to β8 rotated ~90° relative to (D), illustrating a twist of the strands rather than just a simple rotation. (F) View of the lateral gate in BamA, where strand β1 and β16 no longer form a β-sheet interaction to close the barrel; rather, strands β15 and β16 are situated at ~45° angle relative to strand β1. (G) View showing residues along the lateral gate, with β16 sitting tucked inside the barrel and periplasmic loop 7 interacting with strand β1.

Binding of BamCDE leads to a conformational twist of the β-barrel domain of BamA, with the most dramatic change emanating from strand β1 (~45°) and gradually diminishing until strand β9. This conformation is due to the strong interaction of POTRA5 with BamCDE and with periplasmic loop 4, which imposes mechanical strain on the first half of the barrel (Fig. 4, B, D, and E, and movie S2). This leads to a change in the angle of the first eight strands in the membrane, such that strands β1 and β16 no longer associate as a β-sheet, leading to opening of the exit pore along extracellular loops 1, 2, and 3. This agrees with recent studies that have shown that disulfide cross-linking the exit pore in a closed state renders the BAM complex nonfunctional (24). Aligning BamA from our complex structure with the previously reported E. coli BamA structure containing POTRA5 only (PDB ID 4C4V) yielded an RMSD of 1.07 Å for the entire β-barrel domain. However, the RMSD for strands β1 to β8 alone was 2.74 Å, whereas the RMSD for strands β9 to β16 was 0.67 Å, which highlighted the shift observed in our structure.

Although the conformational change of the barrel of BamA led to opening of extracellular loops 1, 2, and 3 along the exit pore, the remaining loops 4, 5, 6, 7, and 8 remained largely unchanged. The exception is that loop 4 undergoes a slight shift to stabilize loop 6 (Fig. 4, C and D). The rest of loop 6 was unchanged, including the conserved VRGF motif (fig. S6). Another consequence of the conformational twist is structural rearrangement at the lateral gate, such that strand β1 no longer interacts with strand β16 (last strand) to close the barrel, in contrast to what has been observed in all other OMPs with known structure (Fig. 4, F and G). Rather, most of strand β16 sits tucked inside the barrel lumen, whereas periplasmic loop 7 and strand β15 contact strand β1 at an offset angle of ~45° (Fig. 4, D to G). This agrees with studies that rendered the BAM complex nonfunctional by disulfide cross-linking the lateral gate closed (24).

Based on our crystal structure of BamACDE and the existing crystal structure of BamAB (14), we report the structure of the fully assembled BAM complex from E. coli (movies S1 and S2 and model S1). As further validation, our structure is in agreement with a model for the BAM complex that we recently reported that was based on all structural, functional, genetics, and biochemical studies to date (34). Our structure reveals that upon binding BamCDE, the barrel domain of BamA undergoes a conformational twist that dramatically changes the angle of the strands (shear) in the membrane, which leads to opening of the exit pore and rearrangement at the lateral gate. This suggests that binding of BamCDE modulates the conformation of BamA, which may serve to regulate the BAM complex. This may also serve to “tune” the β-barrel domain of BamA to fold OMPs with differing shear numbers by adjusting the angle of the strands in the membrane to match that of the substrate OMPs or to further destabilize the local membrane, which would reduce the kinetic barrier for OMP insertion (35). Further, the lumen of the barrel of BamA is fully occluded from the periplasm. Therefore, OMPs are likely inserted into the membrane at the lateral gate rather than first being threaded through the barrel domain. The lateral gate is positioned central to the BAM complex and would be directly accessible for substrate handoff by the accessory lipoproteins or by the POTRA domains of BamA.

Supplementary Materials

www.sciencemag.org/content/351/6269/180/suppl/DC1

Materials and Methods

Figs. S1 to S6

Tables S1 to S6

Movies S1 and S2

References (3641)

REFERENCES AND NOTES

  1. Single-letter abbreviations for the amino acid residues are as follows: A, Ala; C, Cys; D, Asp; E, Glu; F, Phe; G, Gly; H, His; I, Ile; K, Lys; L, Leu; M, Met; N, Asn; P, Pro; Q, Gln; R, Arg; S, Ser; T, Thr; V, Val; W, Trp; and Y, Tyr.
Acknowledgments: We thank H. Bernstein for providing the pJH114 plasmid. J.B. and N.N. are supported by the Department of Biological Sciences at Purdue University and by the National Institute of Allergy and Infectious Diseases (1K22AI113078-01). S.K.B. is supported by the Intramural Research Program of the NIH, National Institute of Diabetes and Digestive and Kidney Diseases. We thank the staff at the Southeast Regional Collaborative Access Team (SER-CAT) beamline at the Advanced Photon Source, Argonne National Laboratory, for their assistance during data collection. Use of the Advanced Photon Source was supported by the Office of Basic Energy Sciences, Office of Science, U.S. Department of Energy, under contract no. W-31-109-Eng-38 (SER-CAT). Coordinates and structure factors for the BamACDE complex have been deposited into the PDB with PDB ID 5EKQ.
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