Abstract
The biogenesis of outer-membrane proteins (OMPs) in gram-negative bacteria involves delivery by periplasmic chaperones to the β-barrel assembly machinery (BAM), which catalyzes OMP insertion into the outer membrane. Here, we examine the effects of membrane thickness, the Escherichia coli periplasmic chaperones Skp and SurA, and BamA, the central subunit of the BAM complex, on the folding kinetics of a model OMP (tOmpA) using fluorescence spectroscopy, native mass spectrometry, and molecular dynamics simulations. We show that prefolded BamA promotes the release of tOmpA from Skp despite the nM affinity of the Skp:tOmpA complex. This activity is located in the BamA β-barrel domain, but is greater when full-length BamA is present, indicating that both the β-barrel and polypeptide transport-associated (POTRA) domains are required for maximal activity. By contrast, SurA is unable to release tOmpA from Skp, providing direct evidence against a sequential chaperone model. By varying lipid acyl chain length in synthetic liposomes we show that BamA has a greater catalytic effect on tOmpA folding in thicker bilayers, suggesting that BAM catalysis involves lowering of the kinetic barrier imposed by the hydrophobic thickness of the membrane. Consistent with this, molecular dynamics simulations reveal that increases in membrane thinning/disorder by the transmembrane domain of BamA is greatest in thicker bilayers. Finally, we demonstrate that cross-linking of the BamA barrel does not affect tOmpA folding kinetics in 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) liposomes, suggesting that lateral gating of the BamA barrel and/or hybrid barrel formation is not required, at least for the assembly of a small 8-stranded OMP in vitro.
Highlights
The outer membranes (OMs) of gram-negative bacteria are densely packed with outer-membrane proteins (OMPs), which are involved in a myriad of functions including the uptake of nutrients, release of waste materials, secretion of virulence factors, and resistance to host defence systems [1]
By varying lipid acyl chain length in synthetic liposomes we show that BamA has a greater catalytic effect on transmembrane domain of OmpA (tOmpA) folding in thicker bilayers, suggesting that barrel assembly machinery (BAM) catalysis involves lowering of the kinetic barrier imposed by the hydrophobic thickness of the membrane
Tryptophan fluorescence emission spectra and SDS-PAGE band shift assays indicated that BamA, the β-barrel domain of BamA (tBamA), and OmpA could be successfully folded into 1,2-diundecanoyl-sn-glycero-3-phosphocholine (DUPC) Large Unilamellar Vesicles (LUVs), following dilution from 8 M urea (Fig. S1a–c), with similar high yields (89.0 ± 2.0%, 88.2 ± 1.2%, and 85.4 ± 1.2%, respectively) (Fig. S2)
Summary
The outer membranes (OMs) of gram-negative bacteria are densely packed with outer-membrane proteins (OMPs), which are involved in a myriad of functions including the uptake of nutrients, release of waste materials, secretion of virulence factors, and resistance to host defence systems [1]. OMPs are synthesized in the cytosol, translocated across the inner membrane, and assisted across the periplasm by chaperones, which prevent their misfolding and aggregation en route to the OM [2]. The final OMP insertion step is mediated by the heteropentameric β-barrel assembly machinery (BAM) complex (BamA-E), by an unknown mechanism [3,4]. The central BAM subunit BamA consists (in Escherichia coli) of a 16-stranded membrane-embedded β-barrel domain preceded by five tandem polypeptide transport-associated (POTRA) domains. BamA is the only BAM complex member for which homologues have been found in all sequenced gram-negative bacterial genomes [5], and BamA-assisted OMP folding has been demonstrated in the absence of other BAM subunits [6,7,8].
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