BamA POTRA Domain Interacts with a Native Lipid Membrane Surface

Abstract

The outer membrane of Gram-negative bacteria is an asymmetric membrane with lipopolysaccharides on the external leaflet and phospholipids on the periplasmic leaflet. This outer membrane contains mainly β-barrel transmembrane proteins and lipidated periplasmic proteins (lipoproteins). The multisubunit protein β-barrel assembly machine (BAM) catalyzes the insertion and folding of the β-barrel proteins into this membrane. In Escherichia coli, the BAM complex consists of five subunits, a core transmembrane β-barrel with a long periplasmic domain (BamA) and four lipoproteins (BamB/C/D/E). The BamA periplasmic domain is composed of five globular subdomains in tandem called POTRA motifs that are key to BAM complex formation and interaction with the substrate β-barrel proteins. The BAM complex is believed to undergo conformational cycling while facilitating insertion of client proteins into the outer membrane. Reports describing variable conformations and dynamics of the periplasmic POTRA domain have been published. Therefore, elucidation of the conformational dynamics of the POTRA domain in full-length BamA is important to understand the function of this molecular complex. Using molecular dynamics simulations, we present evidence that the conformational flexibility of the POTRA domain is modulated by binding to the periplasmic surface of a native lipid membrane. Furthermore, membrane binding of the POTRA domain is compatible with both BamB and BamD binding, suggesting that conformational selection of different POTRA domain conformations may be involved in the mechanism of BAM-facilitated insertion of outer membrane β-barrel proteins.

The Bam complex catalyzes efficient insertion of bacterial outer membrane proteins into membrane vesicles of variable lipid composition

“…Thus, while BamA alone can catalyze limited OMP assembly, our results imply that the Bam complex lipoproteins play an important role in promoting folding in a native lipid context. Although evidence that the BamA POTRA domains interact with negatively-charged lipids (66) and that BamE binds to PG (13,67) suggests that the recruitment of specific lipids stabilizes lipoprotein binding or facilitates OMP folding, our finding that the lipid environment does not significantly affect OMP assembly challenges the hypothesis that the Bam complex functions by exploiting the anionic membrane surface. Likewise, our results indicate that CL, which appears to play a stimulatory role in the assembly of mitochondrial β-barrels (68), does not facilitate Bam complex-mediated folding.…”

Outer Membrane Biogenesis

“…2A). Molecular dynamic (MD) simulations of the BamA in the membrane releveled that all five POTRA domains have high affinity for the membrane, and establish multiple hydrogen bonds with phospholipid head groups and partition directly into the membrane via highly conserved tryptophan residues (25). Partitioning of these tryptophans into the detergent micelle was later observed in the cryo-EM structure of the Bam complex (37).…”

“…This choice of temperature is particularly surprising knowing the low thermostability of the BamA barrel. When MD simulations were repeated with BamA incorporated into the asymmetric lipid bilayer resembling the E. coli OM and under physiological temperatures, no lateral opening was observed (25). …”

“…These mutations restored OMP assembly and allowed growth of the bamE bamB strain at non-permissive temperature. Several of these mutant BamAs also had different mobility on the SDS-PAGE suggesting altered barrel conformation (25). These barrel and eL6 altering mutations, essentially confer a gain of function phenotype bypassing the requirement for BamA communication with the periplasmic lipoproteins.…”

“…However, the exact nature of such a membrane defect in vivo is not understood. Recent MD simulations of the full-length BamA and the cryo-EM structure of the Bam complex in a detergent micelle suggested that Bam complex interactions with the OM are not limited to the BamA barrel domain, but may also involve the POTRA domains and the Bam lipoproteins (25; 37). Though the function of these interactions with the membrane is currently unknown, it is possible that they contribute to the alteration of membrane architecture around the Bam complex.…”