The TPR domain of BepA is required for productive interaction with substrate proteins and the ß-barrel assembly machinery complex.

BepA (formerly YfgC) is an Escherichia coli periplasmic protein consisting of an N-terminal protease domain and a C-terminal tetratricopeptide repeat (TPR) domain. We have previously shown that BepA is a dual functional protein with chaperone-like and proteolytic activities involved in membrane assembly and proteolytic quality control of LptD, a major component of the outer membrane lipopolysaccharide translocon. Intriguingly, BepA can associate with the BAM complex: the ß-barrel assembly machinery (BAM) driving integration of ß-barrel proteins into the outer membrane. However, the molecular mechanism of BepA function and its association with the BAM complex remains unclear. Here, we determined the crystal structure of the BepA TPR domain, which revealed the presence of two subdomains formed by four TPR motifs. Systematic site-directed in vivo photo-cross-linking was used to map the protein-protein interactions mediated by the BepA TPR domain, showing that this domain interacts both with a substrate and with the BAM complex. Mutational analysis indicated that these interactions are important for the BepA functions. These results suggest that the TPR domain plays critical roles in BepA functions through interactions both with substrates and with the BAM complex. Our findings provide insights into the mechanism of biogenesis and quality control of the outer membrane.

Protease homolog BepA (YfgC) promotes assembly and degradation of ß-barrel membrane proteins in Escherichia coli.

Gram-negative bacteria are equipped with quality-control systems for the outer membrane (OM) that sense and cope with defective biogenesis of its components. Accumulation of misfolded outer membrane proteins (OMPs) in Escherichia coli leads to activation of σ(E), an essential alternative σ factor that up-regulates transcription of multiple genes required to preserve OM structure and function. Disruption of bepA (formerly yfgC), a σ(E)-regulated gene encoding a putative periplasmic metalloprotease, sensitizes cells to multiple drugs, suggesting that it may be involved in maintaining OM integrity. However, the specific function of BepA remains unclear. Here, we show that BepA enhances biogenesis of LptD, an essential OMP involved in OM transport and assembly of lipopolysaccharide, by promoting rearrangement of intramolecular disulfide bonds of LptD. In addition, BepA possesses protease activity and is responsible for the degradation of incorrectly folded LptD. In the absence of periplasmic chaperone SurA, BepA also promotes degradation of BamA, the central OMP subunit of the ß-barrel assembly machinery (BAM) complex. Interestingly, defective oxidative folding of LptD caused by bepA disruption was partially suppressed by expression of protease-active site mutants of BepA, suggesting that BepA functions independently of its protease activity. We also show that BepA has genetic and physical interaction with components of the BAM complex. These findings raised the possibility that BepA maintains the integrity of OM both by promoting assembly of OMPs and by proteolytically eliminating OMPs when their correct assembly was compromised.