Structural and functional analysis of the β‐barrel domain of BamA from Escherichia coli

Abstract

In gram-negative bacteria, the assembly of outer membrane proteins (OMPs) requires a β-barrel assembly machinery (BAM) complex, of which BamA is an essential and evolutionarily conserved component. To elucidate the mechanism of BamA-mediated OMP biogenesis, we determined the crystal structure of the C-terminal transmembrane domain of BamA from Escherichia coli (EcBamA) at 2.6 Å resolution. The structure reveals 2 distinct features. First, a portion of the extracellular side of the β barrel is composed of 5 markedly short β strands, and the loops stemming from these β strands form a potential surface cavity, filled by a portion of the L6 loop that includes the conserved VRGF/Y motif found in the Omp85 family. Second, the 4 extracellular loops L3, L4, L6, and L7 of EcBamA form a dome over the barrel, stabilized by a salt-bridge interaction network. Functional data show that hydrophilic-to-hydrophobic mutations of the potential hydrophilic surface cavity and a single Arg547Ala point mutation that may destabilize the dome severely affect the function of EcBamA. Our structure of the EcBamA β barrel and structure-based mutagenesis studies suggest that the transmembrane β strands of OMP substrates may integrate into the outer membrane at the interface of the first and last β strands of the EcBamA barrel, whereas the soluble loops or domains may be transported out of the cell via the hydrophilic surface cavity on dislocation of the VRGF/Y motif of L6. In addition, the dome over the barrel may play an important role in maintaining the efficiency of OMP biogenesis.

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

“…Rather, the glycine likely plays a structural role and is required to produce a stable, folded β-barrel. In fact, crystal structures of the BamA β-barrel indicate that G771 is adjacent to residues F738 and D740, which interact with the highly conserved residue R661 in loop 6 (8,9). These interactions between loop 6 and residues on the inside of the barrel may be important in stabilizing the barrel structure, and we hypothesize that larger amino acids at position 771 may disrupt these interactions.…”

“…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 of Toc75 exhibit chaperone-like function to facilitate import into chloroplasts

“…Although structures have been reported for the orthologs of the Omp85 family, including FhaC, TamA, and BamA, in Gram-negative bacteria (35,36,(43)(44)(45)(46), no structure has been reported for either Sam50 in mitochondria or Toc75 or OEP80 in chloroplasts. Here we report the structure of Toc75, from A. thaliana, consisting of the N-terminal linker and three tandem POTRA domains (Figs.…”

“…Recent X-ray crystallography and cryo-EM studies reported the structures of several Omp85 family members, including FhaC, TamA, and BamA (28,(36)(37)(38)(43)(44)(45)(46)(47). Given the conservation of key features, these structures serve as models for Toc75, albeit with low sequence identity (7%, 7%, and 8%, respectively).…”

Translocation path of a substrate protein through its Omp85 transporter

“…We uncover the critical role of an extracellular b-sheet for FhaC secretion activity. Interestingly, corresponding surface regions of the E. coli Omp85 transporter BamA were also found to be essential for function 33,34 .…”