The Structure of a BamA-BamD Fusion Illuminates the Architecture of the β-Barrel Assembly Machine Core

Abstract

Summary The β-Barrel Assembly Machine (BAM) mediates folding and insertion of integral β-barrel Outer Membrane Proteins (OMPs) in Gram-negative bacteria. Of the five BAM subunits, only BamA and BamD are essential for cell viability. Here we present the crystal structure of a fusion between BamA POTRA4-5 and BamD from Rhodothermus marinus. The POTRA5 domain binds BamD between its TPR repeats 3 and 4. The interface structural elements are conserved in the E. coli proteins, which allowed structure validation by mutagenesis and disulfide crosslinking in E. coli. Furthermore the interface is consistent with previously reported mutations that impair BamA-BamD binding. The structure serves as a linchpin to generate a BAM model where POTRA domains and BamD form an elongated periplasmic ring adjacent to the membrane with a central cavity approximately 30 × 60 Å wide. We propose that nascent OMPs bind this periplasmic ring prior to insertion and folding by BAM.

Flexibility in the Periplasmic Domain of BamA Is Important for Function

“…For example, ≈50% disulfide crosslinking efficiency was observed between cysteines introduced in the first and last strands of the BamA β-barrel (Noinaj et al, 2014) or between cysteines engineered in BamA and BamB (Jansen et al, 2015) or BamA and BamD (Bergal et al, 2016). The data here showed efficient cysteine labeling with maleimide-biotin for the BamA double-cysteine mutants in cells grown in the presence of TCEP and markedly reduced labeling in its absence (Figure 6a).…”

“…The recent reports of BAM complex X-ray structures (Bakelar et al, 2016; Bergal et al, 2016; Chen et al, 2016; Gu et al, 2016) allow us to hypothesize on the role that BamA flexibility plays in BAM function. As seen in Figure 8, BamD interacts with the POTRA5 and the POTRA1–2 domains of BamA forming a ring in the periplasmic side of the BAM complex.…”

“…As seen in Figure 8, BamD interacts with the POTRA5 and the POTRA1–2 domains of BamA forming a ring in the periplasmic side of the BAM complex. This ring is adjacent to the membrane and is thought to bind nascent OMPs (Bakelar et al, 2016; Bergal et al, 2016; Gu et al, 2016; Han et al, 2016). Flexibility at the hinge between POTRA2 and POTRA3 (labeled with an arrow in Figure 8) could then regulate access to the ring and/or modulate its dimensions.…”

“…Instead, it fit best to a population-weighted average of conformations with approximately 25% “compact” and 75% “extended” forms consistent with a model of two rigid subdomains, POTRA1–2 and POTRA3–5, connected by a flexible hinge. It has been proposed that conformational cycling in BamA is necessary for OMP folding and insertion (Rigel et al, 2013), and recent structures of BamA in complex with multiple BAM subunits show that the POTRA domains form a closed ring structure along with BamD, where the ring is adjacent to the membrane (Bakelar et al, 2016; Bergal et al, 2016; Gu et al, 2016; Han et al, 2016). Flexibility at the POTRA2–3 hinge would allow opening and closing of this ring structure, where the ring has been proposed to hold nascent OMPs close to the membrane prior to their insertion and folding.…”

BamA POTRA Domain Interacts with a Native Lipid Membrane Surface

“…A structural understanding of the subunit arrangement in the BAM complex has emerged from several independent xray crystallographic, NMR, and electron microscopy studies of either individual subunits or complexes of fragments of BamA/B, BamA/D, and BamC/D (18,19,21,(23)(24)(25)(26)(33)(34)(35)(36)(37) and recent x-ray structures of the BamA/C/D/E (38,39) and BamA/B/C/D/E complexes (39,40). In addition, the POTRA domains have independently been shown to bind to BamB and BamD (29,32), and it has been suggested that they may template b-strand formation in client OMP polypeptides (23,24,39).…”