Structural characterization of the POTRA domains from A. baumannii reveals new conformations in BamA

Abstract

Recent studies have demonstrated BamA, the central component of the β-barrel assembly machinery (BAM), as an important therapeutic target to combat infections caused by Acinetobacter baumannii and other Gram-negative pathogens. Homology modeling indicates BamA in A. baumannii consists of five polypeptide transport-associated (POTRA) domains and a β-barrel membrane domain. We characterized the POTRA domains of BamA from A. baumannii in solution using size-exclusion chromatography small angle X-ray scattering (SEC-SAXS) analysis and determined crystal structures in two conformational states that are drastically different than those previously observed in BamA from other bacteria, indicating that the POTRA domains are even more conformationally dynamic than has been observed previously. Molecular dynamics simulations of the POTRA domains from A. baumannii and Escherichia coli allowed us to identify key structural features that contribute to the observed novel states. Together, these studies expand on our current understanding of the conformational plasticity within BamA across differing bacterial species.

Keywords: Gram-negative bacteria; membrane protein; multidrug resistance; protein folding; structural biology.

Figure 1.. Sequence comparison of POTRAs 1–4 and purification of this domain from A. baumannii.

A. Alignment of the AbBamA POTRA 1–4 amino acid sequence with other structurally characterized orthologs of BamA. The secondary structure of BamA POTRA 1–4 from A. baumannii is shown based on our studies here. The boundaries for each POTRA domain are indicated in blue, purple, green, and brown. B. The SEC profile from native and selenomethionine substituted (panel C) AbBamA POTRA 1–4 showing a pure sample running at ~40 kDa (expected mass is 41 kDa including the His tag).

Figure 2.. Structure determination of AbBamA POTRA 1–4.

A. AbBamA POTRA 1–4 crystals showing small thin finely stacked plates. B. The structure of AbBamA POTRA 1–4 highlighting the selenomethionine sites used for phasing. C. Representative electron density (blue isosurface; 1 σ) for the AbBamA POTRA 1–4 structure. D. The asymmetric unit of the structure in space group P3₂21 showing two molecules with pseudo two-fold non-crystallographic symmetry. Orthogonal views are shown.

Figure 3.. The structures of AbBamA POTRA 1–4 in condensed and extended conformational states.

A. The assembly of AbBamA POTRA 1–4 in the asymmetric unit in space group P3₂21 and P1 (panel B), both with pseudo two-fold non-crystallographic symmetry. C. A superposition of the two chains in space group P3₂21 (RMSD value of 3.6 Å) and P1 (panel D; RMSD value of.7 Å). The conformation of the structure in P1 is more extended. E. A superposition of chains A (RMSD of 6.3 Å) and B (panel F; RMSD of 8.5 Å) from each respective space group.

Figure 4.. SAXS analysis of AbBamA POTRA 1–4.

A. An overall superposition of chain A from space group P1 to both chains in space group P3₂21. Orthogonal views are shown. B. The same structural alignment as in panel A, yet superimposed along POTRAs 3 and 4, demonstrating a large ~45° swing of POTRA 1 and 2 moving ~54 Å. C. SEC-SAXS analysis of AbBamA POTRA 1–4 gave an R_g of 35 Å, an average mass of 41.5 kDa, and a D_max value of 126 Å (panel D). E. A Kratky plot is consistent with a globular fold containing flexible linkers on its termini.

Figure 5.. AbBamA POTRA 1–4 reveals new conformations in BamA.

A. A structural alignment of POTRA 1–4 from AbBamA with EcBamA alone (PDB ID 2QCZ, 2QDF, and 3EFC) and EcBamA within the BAM complex (PDB ID 5EKQ). The left panel is the same view as in Fig 4. panels A and B, while the top-right view is rotated ~45° from the left view, with the right -bottom being orthogonal to the top-right. B. CRYSOL was used to compare calculated scattering curves of the structures from panel A to the experimental scattering curve in panel Fig. 4C. χ² values indicate the closest match is chain A from space group P1 of AbBamA POTRA 1–4 (pink; χ² value of 1.3), while the structures for EcBamA have χ² values ranging from 1.6 – 7.2; NgBamA has a χ² value of 1.6. SREFLEX was used to improve the fit of chain A from space group P1 of AbBamA POTRA 1–4, producing UC01 (red), which gave a χ² value of 1.1. C. A superposition of chain A from space group P1 of AbBamA POTRA 1–4 (pink) and UC01 (red) with the DAMMIN/IF SAXS envelope. D. Modeling the new conformations with the full length E. coli BamA structure (PDB ID 5D0Q). E. A surface depiction of the two conformations from panel D, where the POTRA domains are in the periplasm and at the membrane surface.

Figure 6.. The conformation of AbBamA and EcBamA POTRA 1–4 in MD simulations.

AbBamA POTRAs 1,2 and 4 are shown in dark grey, and POTRA3 is in yellow. EcBamA POTRAs 1,2 and 4 are shown in blue and POTRA3 is in orange. A. The initial conformation for AbBamA and EcBamA POTRAs. B,C. Snapshots of “open” (B) and “closed” states (C) of AbBamA. E,F. Snapshots of “open” (B) and “closed” states (C) of EcBamA. D,G. Overlapping conformations of AbBamA (D) and EcBamA (G) chain A during the trajectories, with one snapshot every 20 ns. Structures are aligned based on POTRA3 β-sheet structures in panels A, D, and G. H,I. Distribution of angles formed by different POTRA domains of AbBamA (H) and EcBamA (I).