Structural basis for substrate selection by the translocation and assembly module of the β-barrel assembly machinery

Abstract

The assembly of proteins into bacterial outer membranes is a key cellular process that we are only beginning to understand, mediated by the β-barrel assembly machinery (BAM). Two crucial elements of that machinery are the core BAM complex and the translocation and assembly module (TAM), with each containing a member of the Omp85 superfamily of proteins: BamA in the BAM complex, TamA in the TAM. Here, we used the substrate protein FimD as a model to assess the selectivity of substrate interactions for the TAM relative to those of the BAM complex. A peptide scan revealed that TamA and BamA bind the β-strands of FimD, and do so selectively. Chemical cross-linking and molecular dynamics are consistent with this interaction taking place between the first and last strand of the TamA barrel domain, providing the first experimental evidence of a lateral gate in TamA: a structural element implicated in membrane protein assembly. We suggest that the lateral gates in TamA and BamA provide different environments for substrates to engage, with the differences observed here beginning to address how the TAM can be more effective than the BAM complex in the folding of some substrate proteins.

Figure 1. Structural comparison of BamA and TamA

A. Schematic of the BAM complex and the TAM involved in outer membrane protein assembly. In E. coli, the BAM complex contains five subunits (pdb 5d0o) and the TAM contains two subunits, TamA and TamB (TamA crystal structure pdb 4c00). No crystal structure is available for TamB, but its elongate shape has been established by atomic force microscopy (Shen et al., 2014). B. Structural alignment of BamA (pdb 5d0o chain A, aqua) and TamA (pdb 4c00, purple). The different shear numbers for barrel domains of TamA (S=20) and BamA (S=22) (Noinaj et al., 2015) are distinguishable in the overlay, as the β-strands in the TamA barrel domain are closer to being perpendicular to the membrane. C. According to current definitions (Noinaj et al 2015), the crystal structure of TamA is in a tucked conformation, with β-strand 16 spanning five residues (Q⁵⁶⁸ to G⁵⁷²). D. The barrel domains of TamA (pdb 4c00) and BamA (pdb 4c4v) were rendered and the surface area of each was calculated in the PyMOL software package(Schrodinger, 2010). The surface areas are documented in Ų. E. Comparison of the TamA (pdb 4c00) and BamA (pdb 4c4v) barrel lumen. Each barrel domain is divided in half to examine the amino acid composition inside the barrel lumen. Internal facing residues that are positively charged are shown in blue and negatively charged in red.

Figure 2. Characterization of the BamA-TamA chimeras in BamA shutdown strain

A. The constructs as cartoons. B. Plasmid-borne production of BamA, BamA_POTRA:TamA_BARREL, TamA_POTRA:BamA_BARREL and TamA in the bamA shutdown strain (see Supplementary Fig. S1). Following a four-hour depletion, cells were sub-cultured in fresh LB media supplemented with glucose and after three hours of further bamA repression, total membrane extracts were prepared and analyzed by BN-PAGE. Filters were probed with anti-sera against BamB, BamC, BamD, BamE or the POTRA domains of BamA. The position of the BAM complex is shown, it runs in two conformational states of similar but not identical size, but each having all five subunits. The position of the BamAB module is shown. C. Production of BamA, BamA_POTRA:TamA_BARREL, TamA_POTRA:BamA_BARREL and TamA in a ΔtamA strain. Total membrane extracts were prepared as in B and analyzed by BN-PAGE. Filters were probed with anti-sera raised against the POTRA domains of TamA, or against TamB. The position of the TAM is shown and the population of free TamA is indicated (see Supplementary Fig. S2).

Figure 3. Effects on substrate assembly in ΔtamA complemented with BamA-TamA chimeras

FimD assembly was monitored over time in ΔtamA cells harbouring the indicated plasmids, as assessed by pulse chase analysis. Aliquots were taken at 10 sec, 2, 4, 8, 16 and 32 min, treated with proteinase K (+/− PK). Total protein was analyzed by SDS-PAGE and storage phosphor-imaging. The time increment is indicated as a graded triangle above the autoradiogram and a cartoon of the relevant β-barrel species expressed are indicated on the right. The position of FimD and its fragments A, B and C are indicated at right. A is the N-terminal 50 kDa fragment, C is the C-terminal 40kDa fragment, while B is a central fragment of an assembly intermediate of FimD that accumulates in the absence of the TAM (Stubenrauch et al., 2016b).

Figure 4. Dissection of BamA and TamA substrate selection

A. Topology map representation of the 24 β-strands (Table 1) and extramembrane domains of the outer membrane usher protein FimD. The structure of FimD (pdb 3RFZ, chain B) is color-coded accordingly, with N- and C-termini indicated. B. FimD peptide blot assay containing individual peptides that represent each β-strand of the barrel domain (1 through to 24). This β-strand peptide display was probed with recombinant OmpF to guage any non-specific binding (see Methods). C. Recombinant BamA or TamA were used to probe the β-strand peptide display. D. FimD peptide blots were also probed with truncated, domain-only versions of BamA and TamA as indicated.

Figure 5. FimD peptide binding to TamA

A. The FimD peptide was incubated with purified TamA and binding assessed by chemical crosslinking with BS3. Coomassie stained SDS-PAGE shows TamA purified protein cross-linking and the western blot is probed with anti-strep to confirm it cross-links to the FimD Peptide. B. Structure of TamA (pdb 4c00) mapping the position of the tryptic digest peptide identified as cross-linked to FimD peptide (silver). C–E. The cross-linked Lys²⁸³ in TamA is depicted by representing its sidechain that points into the lumen of the barrel domain. Selected states of the barrel domain of TamA (purple) from MD simulations, with the FimD peptide shown in yellow. C. TamA with free peptide at the point of closest approach between the C-terminal Lys of the peptide and Lys²⁸³ of TamA (see also Supplementary Fig. S5). D. TamA with peptide in the lateral gate at the point of closest approach (see also Supplementary Fig. S6). E. TamA with the peptide cross-linked to Lys²⁸³ via BS3. Three hydrogen bonds between the cross-linker and TamA’s first β-strand are shown in light blue (see also Supplementary Fig. S8).

Figure 6. PMFs of lateral gate separation

Potential of mean force (PMF) as a function of separation between β-strands 1 and 16 for TamA (green), BamA (blue), and FhaC (red). The energy required to separate the strands is similar for BamA and TamA (5 to 10 kcal/mol), and both are much lower than the control protein FhaC (25 to 35 kcal/mol).