The structure of the β-barrel assembly machinery complex

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.

Fig. 1. The structure of the BAM complex

A. The pathway for the biogenesis of β-barrel outer membrane proteins 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 90º 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 strand β1-β8, which can be seen here. This panel is rotated ~90º along the y-axis relative to the top view of panel B. The overall measurements of the BAM complex are ~115 × 115 × 115 Å.

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 90º 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 90º 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 Ų, containing a mix of hydrogen bonds, salt bridges, and hydrophobic interactions. D. A view from the top which is rotated 90º along the x-axis to further illustrate the extensive binding interface.

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, while 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 ~1,100 Ų, containing a mix of hydrogen bonds, salt bridges, and hydrophobic interactions. Residue R197 of BamD clearly be 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. While minimal here, these interactions 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 Ų, 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.

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

A. Superposition of BamA from our structure (green) with PDB ID 4C4V (cyan) reveals ~45º hinge-like 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-β8 with the most dramatic change emanating from strand β1 (~45º) and gradually diminishing until strand β9 (Movie S2). This leads to opening of the exit pore 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-β8 of the barrel domain of BamA. E. View of the strand shift along strandsβ1-β8 rotated ~90º relative to panel 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 ~45º 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.