Crystal Structure of BamD: An Essential Component of the β-Barrel Assembly Machinery of Gram-Negative Bacteria

Abstract

Folding and insertion of integral β-barrel proteins in the outer membrane is an essential process for Gram-negative bacteria that requires the β-barrel Assembly Machinery (BAM). Efficient OMP folding and insertion appears to require a consensus C-terminal signal in OMPs characterized by terminal F or W residues. The BAM complex is embedded in the outer membrane and, in E. coli, consists of the β-barrel BamA and four lipoproteins BamBCDE. BamA and BamD are broadly distributed across all species of Gram-negative bacteria whereas the other components are present in only a subset of species. BamA and BamD are also essential for viability suggesting that these two proteins constitute the functional core of the bacterial BAM complex. Here we present the crystal structure of BamD from the thermophilic bacteria Rhodothermus marinus refined to 2.15 Å resolution. The protein contains 5 tetratricopeptide repeats (TPRs) organized into two offset tandems, each capped by a terminal helix. The N-terminal domain contains three TPRs and displays remarkable structural similarity with proteins that recognize targeting signals in extended conformations. The C-terminal domain harbors the remaining 2 TPRs and previously described mutations that impair binding to other BAM components map to this domain. Therefore, the structure suggests a model where the C-terminal domain provides a scaffold for interaction with BAM components, while the N-terminal domain participates in interaction with the substrates, either recognizing the C-terminal consensus sequence or binding unfolded OMP intermediates.

Inhibition of the β-barrel assembly machine by a peptide that binds BamD

“…However, this conclusion does not imply that each of the >50 different OMPs present in the OM specifically requires BamD for its assembly (39). Other components of the Bam complex also interact with substrates (13,15,(19)(20)(21)(22)38) and may have functions that overlap with that of BamD such that certain OMPs can be assembled in its absence. Rather, we suggest that BamD's essentiality in substrate binding reflects a kinetic effect on In the in vitro experiments, full-length BamA substrates containing mutations in the β-signal sequence were denatured and then diluted into solutions containing 0.5% lauryldimethylamine-N-oxide (LDAO) or BamABCDE proteoliposomes to assess, respectively, the effects of the mutations on the stability of the folded β-barrel and on the folding mechanism carried out by the Bam complex.…”

“…Sam35 and BamD share no obvious sequence homology but perform the same receptor-like function in binding unfolded substrates at the surface of the membrane into which those substrates are inserted (21,30); therefore, the conservation of the β-barrel assembly mechanism extends beyond the steps performed by BamA. Previous reports demonstrating that some mitochondrial β-barrels can be assembled in bacteria and that some bacterial OMPs can be assembled in mitochondria implied that the assembly complexes have similar substrate recognition mechanisms (40)(41)(42)(43), and structural studies (13)(14)(15)(44)(45)(46) suggested that BamD might recognize OMP substrates based on its homology to proteins that bind C-terminal peptides (e.g., a component of the mitochondrial translocase, Tom70; a peroxisomal protein receptor, PEX5; and a factor responsible for organizing Hsp chaperone proteins, Hop). Our results connect these observations and provide the underlying molecular basis for substrate recognition, which explains the reciprocity of β-barrel assembly between mitochondria and bacteria.…”

“…However, the mechanism by which β-barrel assembly proceeds is only beginning to be elucidated. Structures of the components of the bacterial β-barrel assembly machine (BamA-E) have been determined recently, and several hypotheses about how they facilitate the assembly of its substrates have been proposed (7)(8)(9)(10)(11)(12)(13)(14)(15)(16)(17). The β-barrel component BamA contains a kinked β-strand, which might allow substrate proteins to be inserted by passing laterally from the lumen of the BamA β-barrel into the hydrophobic membrane (18).…”

“…The POTRA domains bind the other four components of the assembly complex, BamB-E, which are lipoproteins. Structural and biochemical studies have suggested that the POTRA domains, BamB, and BamD may interact with substrate proteins during their assembly (13,15,(19)(20)(21)(22). However, more detailed information is required about the nature of those interactions and whether they are critical in the assembly process to develop an integrated mechanistic model.…”

A mortise–tenon joint in the transmembrane domain modulates autotransporter assembly into bacterial outer membranes

“…20) and discrete motifs 21 in the sequences of autotransporters that have been conserved through evolution. One conserved motif referred to as 'the b-motif' 21 , appears to act as targeting sequence recognized by the BAM complex [22][23][24][25][26] . A second conserved 'a-linker motif' was found to be situated within the lumen of several autotransporter barrel domains for which crystal structures are available 21 .…”