Effects of Periplasmic Chaperones and Membrane Thickness on BamA-Catalyzed Outer-Membrane Protein Folding

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.

Identification of a novel post‐insertion step in the assembly of a bacterial outer membrane protein

“…In an alternative model, it has been proposed that the Bam complex facilitates the membrane integration of folded or partially folded β barrels by perturbing the lipid bilayer (Noinaj et al ., 2013 ). This model is supported by evidence that at least some OMPs begin to fold prior to membrane integration (Ieva et al ., 2008 ;Lee et al ., 2016 ;Sikdar et al ., 2017 ), that BamA lowers the kinetic barrier for OMP insertion imposed by lipid head groups (Gessmann et al ., 2014 ), that BamA exerts a greater catalytic effect on OMP folding in thicker bilayers (Schiffrin et al ., 2017 ), and that BamA is highly dynamic in liposomes (Doerner and Sousa, 2017 ). The crystal structure of the Bam complex shows that the POTRA domains and the four lipoproteins form a ring below the BamA β barrel.…”

Inter-domain dynamics in the chaperone SurA and multi-site binding to its unfolded outer membrane protein clients

“…SurA binds unfolded OmpX with low affinity (K d,app of ~800 nM), as measured by microscale thermophoresis (MST) ( Supplementary Fig. 8), similar to the affinity of SurA for other OMPs 28,43,44 . SurA-OmpX complexes were assembled by rapid dilution of urea-denatured OmpX into a solution of SurA (final concentrations: 5 µM OmpX, 5 µM SurA, 0.24 M urea) (see Methods) immediately prior to crosslinking with DSBU.…”

“…Despite its key role in OMP biogenesis and bacterial virulence 51,52 , how SurA binds its OMP substrates both specifically, but weakly 28,44,49 , and how it is able to protect its clients from aggregation and deliver them to BAM for folding into the OM, remain poorly understood in molecular detail. Previous NMR studies have shown that OmpX, tOmpA and FhuA are dynamically disordered when bound to SurA 40 -42 .…”

High affinity of Skp to OmpC revealed by single-molecule detection

“…Our results revealed that the free energy of Skp3 binding to OmpC is -20 kcal·mol -1 , which supports the ideas that Skp3 is a superior holdase 17 and de-aggregation agency 6 to OMPs. Since the binding energy is so large, the release of the substrates from Skp may need the help of LPS 35,36 , DegP 2,37 or BAM system 38,39 , ensuring the direction of OMPs transportation towards the final destination.…”