The Lipid Bilayer-Inserted Membrane Protein BamA of Escherichia coli Facilitates Insertion and Folding of Outer Membrane Protein A from Its Complex with Skp

Abstract

Folding of β-barrel membrane proteins, either from a urea-unfolded form or from chaperone-bound aqueous forms, has been characterized for pure lipid bilayers. The impact of preinserted integral proteins from biomembranes has not been examined in biophysical comparisons, but this knowledge is important for the characterization of protein assembly machinery in membranes to distinguish specific effects from unspecific effects. Here, folding was studied for a β-barrel membrane protein, outer membrane protein A (OmpA) from Escherichia coli, in the absence and presence of two other preinserted integral proteins, BamA of the β-barrel assembly machinery complex (BAM) from E. coli and FomA from Fusobacterium nucleatum. Three different preformed lipid membranes of phosphatidylcholine were prepared to compare the folding kinetics of OmpA, namely, proteoliposomes containing either BamA or FomA and pure liposomes. Urea-unfolded OmpA folded faster into phosphatidylcholine bilayers containing FomA than into pure lipid bilayers, but the kinetics of OmpA folding and insertion were fastest for bilayers containing BamA. Incorporation of BamA into lipid bilayers composed of phosphatidylcholine and phosphatidylethanolamine greatly weakened the inhibiting effect of phosphatidylethanolamine on the folding of OmpA. Folding of OmpA from its complex with the periplasmic chaperone Skp into bilayers composed of phosphatidylethanolamine and phosphatidylcholine was inhibited in the absence of BamA but facilitated when BamA was present, indicating an interaction of Skp-OmpA complexes with BamA.

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

“…Previous studies have shown that BamA alone accelerates the folding of OmpA and other empty b-barrel proteins including OmpX and OmpLA into LUVs that contain PG or a small amount of PE (20%), but only at high pH (34,36). Furthermore, even in the presence of BamA, the assembly of OMPs into liposomes composed purely of neutral PC lipids was faster and more efficient, and BamA relieved the inhibitory effect of lipid head groups on the assembly of some proteins more effectively than others (34).…”

“…In the second assay we evaluated the orientation of the Bam complex using a tryptic digest. Previous work has shown that when BamA molecules insert into liposomes in an inside-out orientation (i.e., with their POTRA domains exposed on the surface), trypsin treatment generates a ~43 kD species (BamA*) that corresponds to the transmembrane β-barrel (36). We also expected that trypsin would digest Bam complex lipoproteins that are bound to surface exposed POTRA domains.…”

“…Alternatively, by distorting the lipid bilayer, the Bam complex might promote the integration of OMPs based on their intrinsic thermodynamic properties. Consistent with the possibility that the Bam complex directly overcomes inhibitory effects exerted by lipids, BamA alone accelerates the folding of at least some OMPs into liposomes containing small amounts of native head groups (34,36) as well as relatively thick bilayers that often inhibit spontaneous assembly (19). More significantly, in the presence of SurA, the Bam complex has been shown to catalyze the assembly of OMPs into proteoliposomes that contain native E. coli lipids near neutral pH and within roughly physiological timescales (37)(38)(39)(40).…”

“…In one assay we exploited the observation that properly folded BamA, like many other folded OMPs, migrates more rapidly than its predicted molecular weight on SDS-PAGE in the absence of heat (16,39,45). The so-called "heat modifiability" of BamA is only observed on gels that are run at 4 o C (36,39). In the second assay we evaluated the orientation of the Bam complex using a tryptic digest.…”

“…In one set of experiments, we monitored the ability of the Bam complex to catalyze the folding of OmpA, a model protein that has been studied extensively both in vivo and in vitro (25,36). OmpA is comprised of an N-terminal 8-stranded β-barrel and an independently folded C-terminal periplasmic domain.…”