The sacrificial adaptor protein Skp functions to remove stalled substrates from the β-barrel assembly machine

Abstract

The biogenesis of integral β-barrel outer membrane proteins (OMPs) in gram-negative bacteria requires transport by molecular chaperones across the aqueous periplasmic space. Owing in part to the extensive functional redundancy within the periplasmic chaperone network, specific roles for molecular chaperones in OMP quality control and assembly have remained largely elusive. Here, by deliberately perturbing the OMP assembly process through use of multiple folding-defective substrates, we have identified a role for the periplasmic chaperone Skp in ensuring efficient folding of OMPs by the β-barrel assembly machine (Bam) complex. We find that β-barrel substrates that fail to integrate into the membrane in a timely manner are removed from the Bam complex by Skp, thereby allowing for clearance of stalled Bam–OMP complexes. Following the displacement of OMPs from the assembly machinery, Skp subsequently serves as a sacrificial adaptor protein to directly facilitate the degradation of defective OMP substrates by the periplasmic protease DegP. We conclude that Skp acts to ensure efficient β-barrel folding by directly mediating the displacement and degradation of assembly-compromised OMP substrates from the Bam complex.

FkpA Enhances Membrane Protein Folding using an Extensive Interaction Surface

“…It is also possible that FkpA-uOMP complexes have the capacity to be fully degraded by DegP as has recently been shown for tightly-bound Skp-uOMP complexes (Figure 7). 67 Previous work has suggested that the chaperone function of FkpA is localized to either the N-terminal dimerization 26,27 or the C-terminal PPIase domain, 24,28 but we find that neither domain alone exhibits chaperone function nor high affinity uOmpA171 binding. Some binding of uOmpA171 to N-FkpA was observed even though it was 10-fold weaker than the binding of the full-length protein, which may explain why N-FkpA prevented protein aggregation in some in vitro assays but was unable to rescue periplasmic stress when introduced into a ΔfkpA ΔdegP deletion strain.…”

The Escherichia coli outer membrane protein OmpA acquires secondary structure prior to its integration into the membrane

“…S5C and S5D). Based on the observation that the elimination of the periplasmic chaperone Skp reduces the degradation of OMPs that contain β signal mutations and enhances their assembly (13,14), we next wished to see if the level of the split OmpA mutants would increase in strain XW102 (MC4100 ΔompA J o u r n a l P r e -p r o o f Δskp). Interestingly, the assembly of the single mutants was almost completely restored, while the folded form of the double mutant became detectable (Figs.…”

“…Skp is a jellyfish-like homotrimer that can accommodate unfolded or partially folded OMPs in its central cavity to protect them from misfolding in the aqueous environment ( 11 , 12 ). Skp either transfers OMPs to other chaperones such as SurA or identifies defective OMPs and directs them to proteases for degradation ( 13 , 14 ). SurA targets OMPs to a heteroligomer known as the b arrel a ssembly m achinery (Bam) complex that catalyzes their insertion into the OM.…”

The periplasmic chaperone Skp prevents misfolding of the secretory lipase A from Pseudomonas aeruginosa

“…Although no OMP essentially depends on Skp in the presence of SurA ( Denoncin et al, 2012 ; He & Hiller, 2018 ), other moonlighting activities have been suggested exclusively for Skp. Recently, the chaperone has been described as an adaptor protein that delivers the misfolded OMP LamB to the periplasmic protease DegP ( Combs & Silhavy, 2022 ). Skp is a potent disaggregase of OmpC and OmpX ( Li et al, 2018 ; Chamachi et al, 2022 ) and it also demonstrates the holdase and disaggregase activity towards several soluble proteins ( Purdy et al, 2007 ; Wagner et al, 2009 ; Entzminger et al, 2012 ).…”