Gram-negative outer membrane proteins (OMPs) are characterized by a transmembrane domain with a ß-barrel structure. OMPs contribute to retaining the outer membrane (OM) barrier and add to various functions of the OM including nutrient transport, host-pathogen interaction, or toxin export. After the endosymbiotic events, OMPs have been conserved in the mitochondrial and chloroplast OMs as essential proteins. The essential process of biosynthesis of the ß-barrel membrane proteins is insertion into the OM with proper folding termed assembly. Assembly is mediated by a molecular machine containing highly conserved Omp85 superfamily protein. In E. coli, Omp85 superfamily protein, BamA builds the BAM complex with four different lipoproteins, BamB, BamC, BamD, and BamE. BamA and BamD are essential for E. coli viability. BamA is itself a ß-barrel membrane protein, and between the first and last strand of BamA is the catalytic site for OMPs assembly termed lateral gate. The current model proposed that the lateral gate recognizes the ß-signal which is the specific amino-acid sequence within the most C-terminal ß-strand of substrate proteins and then initiates the assembly reaction. However, the interaction between substrate and machinery during assembly remains unclear.
To seek further regions containing affinity to the machinery, we employed a peptidomimetic competition assay based on EMM assembly assay which is in vitro reconstitution assay using isolated E. coli membrane fraction. Peptides were systematically designed from the major E. coli OMP OmpC. In addition to the ß-signal, OmpC contained several regions with affinity to the BAM complex. These regions contained ß-signal-like sequences with the same orientation strands as the last strand, indicating that OMPs contained multiple ß-signals. Surprisingly, multiple ß-signal rule which repeats the ß-signal at several strands at the C-terminal side of the OMPs were conserved not in bacteria but in mitochondria. We identified that BamD was the receptor of the multiple ß-signals in E. coli. BamD stimulated the folding of multiple ß-signal-containing regions by recognizing multiple ß-signal of the substrate with distinct regions of BamD. This folding acceleration mechanism contributed to retaining OM integrity. Therefore, our findings provide an efficient antibiotic target for protein assembly of gram-negative bacteria.