Characterization of a stalled complex on the β-barrel assembly machine

Abstract

The assembly of β-barrel proteins into membranes is mediated by an evolutionarily conserved machine. This process is poorly understood because no stable partially folded barrel substrates have been characterized. Here, we slowed the folding of the Escherichia coli β-barrel protein, LptD, with its lipoprotein plug, LptE. We identified a late-stage intermediate in which LptD is folded around LptE, and both components interact with the two essential β-barrel assembly machine (Bam) components, BamA and BamD. We propose a model in which BamA and BamD act in concert to catalyze folding, with the final step in the process involving closure of the ends of the barrel with release from the Bam components. Because BamD and LptE are both soluble proteins, the simplest model consistent with these findings is that barrel folding by the Bam complex begins in the periplasm at the membrane interface.outer membrane | Bam complex | β-barrel | protein folding T he assembly of β-barrel membrane proteins into the outer membrane (OM) of Gram-negative bacteria, mitochondria, and chloroplasts is facilitated by conserved cellular machinery (1-4). The β-barrel assembly machine (Bam) folds and inserts integral membrane proteins into the OM of Gram-negative organisms (5). Bam is a five-protein complex consisting of the essential protein BamA, a β-barrel itself, and four lipoproteins, BamB, -C, -D, and -E, of which only BamD is essential (4-8). The Bam complex recognizes a large number of different substrates, but how each component catalyzes the folding and insertion of such structurally diverse substrates is unclear.How β-barrels are assembled into membranes is not obvious. Where and how folding occurs is unclear because intermediates could contain both exposed polar amides and hydrophobic residues until the barrel has completed its fully hydrophobic exterior. By contrast, α-helical membrane proteins have internally satisfied hydrogen bonds, making stepwise assembly from stable secondary structural elements possible. Although Bam has been shown to accelerate membrane β-barrel assembly (9-11), the transient nature of folding intermediates has made accumulating such discrete species for characterization difficult (12-15). If structurally defined folding intermediates were to exist long enough for characterization, they could reveal crucial aspects of the folding process.Here, we studied the assembly of an essential, slow-folding β-barrel, LptD. LptD is one of two components of the OM translocon that transports lipopolysaccharide to the cell surface (16-18). The other component, LptE, is a lipoprotein that forms a plug inside the LptD barrel (19)(20)(21)(22). LptD also contains two disulfide bonds (23), and its assembly involves the formation of consecutive disulfide bonds that after barrel folding rearrange to form nonconsecutive disulfide bonds (24). The assembly of LptD is orders-ofmagnitude slower (∼20 min versus seconds) than that of other barrel substrates (24-26). Because of the slow rate of folding and our ability to use oxidation sta...

Redefining the essential trafficking pathway for outer membrane lipoproteins

“…The Bam machine has two essential OMP substrates: the LPS insertase LptD and BamA. LptD is the cell's most complicated OMP substrate: its folding requires both assistance from an additional essential OM lipoprotein (LptE) and disulfide bond rearrangement to achieve the oxidized, functional form (LptD OX ) (41,42). We found that cpxA24 Δlpp ΔrcsB cells lacking lolB produced the same levels of both BamA and LptD OX as lolB + controls (Fig.…”

Outer Membrane Biogenesis

“…Ability to recognize the substrates is an essential function of BamD, and the peptides containing the β signal, which bind BamD, are toxic because they inhibit β barrel assembly (34). However, the function of BamD is not limited to the initial substrate recognition, because BamD remains closely associated with the substrate throughout entire OMP assembly and must play an active role during this process (40; 56). One of these roles is to regulate BamA conformational cycling through the direct interaction with P5 (see BamA conformational mobility section for more details) (75; 76).…”

“…The rearrangement of [1,2] oxidized LptD into the mature form with non-consecutive disulfide bonds ([1,3][2,4]-LptD) happens after [1,2] LptD is folded around LptE, which brings C-terminal Cys residues in proximity to N-terminal Cys residues (11). Accordingly, conditions that limit LptE stall LptD folding and LptD accumulates in the [1,2] form (56). In addition, when LptE is limiting the [1,2] form of LptD does not accumulate on the Bam complex, demonstrating that LptE is recognized by the Bam complex first, and suggesting that LptE is required not only for LptD folding but also for a recognition of LptD by the Bam complex (56).…”

“…Accordingly, conditions that limit LptE stall LptD folding and LptD accumulates in the [1,2] form (56). In addition, when LptE is limiting the [1,2] form of LptD does not accumulate on the Bam complex, demonstrating that LptE is recognized by the Bam complex first, and suggesting that LptE is required not only for LptD folding but also for a recognition of LptD by the Bam complex (56). …”

“…One such study concerned an assembly-defective mutant of lptD , lptD4213 (56). lptD4213 is 23 amino acid deletion in eL4, and this mutant was instrumental to the discovery of the Bam complex (7; 20; 80; 81; 94).…”