Chaperones Skp and SurA dynamically expand unfolded OmpX and synergistically disassemble oligomeric aggregates

Abstract

Periplasmic chaperones 17-kilodalton protein (Skp) and survival factor A (SurA) are essential players in outer membrane protein (OMP) biogenesis. They prevent unfolded OMPs from misfolding during their passage through the periplasmic space and aid in the disassembly of OMP aggregates under cellular stress conditions. However, functionally important links between interaction mechanisms, structural dynamics, and energetics that underpin both Skp and SurA associations with OMPs have remained largely unresolved. Here, using single-molecule fluorescence spectroscopy, we dissect the conformational dynamics and thermodynamics of Skp and SurA binding to unfolded OmpX and explore their disaggregase activities. We show that both chaperones expand unfolded OmpX distinctly and induce microsecond chain reconfigurations in the client OMP structure. We further reveal that Skp and SurA bind their substrate in a fine-tuned thermodynamic process via enthalpy–entropy compensation. Finally, we observed synergistic activity of both chaperones in the disaggregation of oligomeric OmpX aggregates. Our findings provide an intimate view into the multifaceted functionalities of Skp and SurA and the fine-tuned balance between conformational flexibility and underlying energetics in aiding chaperone action during OMP biogenesis.

Dynamic interplay between the periplasmic chaperone SurA and the BAM complex in outer membrane protein folding

“…OMPs bind in a cradle formed by the Core and P1 domains, with binding sites primarily located on the core domain 30 , 31 . OMP binding to SurA causes the OMP to populate expanded states 31 , 34 , 35 The OMP–SurA complex binds to BAM, to form an OMP–SurA–BAM ternary complex, resulting in conformational changes in both SurA (opening of the P1 and P2 domains) and BAM (favouring the lateral-closed state, which we propose to be the OMP acceptor state). The OMP binding cradle of SurA is oriented into the BAM periplasmic ring, allowing release of the unfolded OMP into a protective ‘chaperonin-like’ environment 5 , and presentation of the OMP to the BamA β-barrel for folding in a C- to N-terminus direction via β-strand elongation from β1 of the BamA barrel 5 , 52 , 53 , 59 , 61 , 114 .…”

“…Together, our results enable a model to be presented of how SurA binds its clients, and delivers them to BAM for folding and insertion into the OM. SurA is known to bind and prevent OMPs from aggregation, and also to modulate the conformational landscape of unfolded OMPs to aid folding 103 , most likely via the creation of extended states in their clients 5 , 31 , 34 , 35 . Here we propose that SurA, as part of the ‘donor’–substrate complex (SurA–OMP), binds directly to BAM, shifts the equilibrium position of BAM towards the lateral-closed (‘inward-open’) acceptor state, to allow the incoming OMP access to the active BamA catalyst (Fig.…”

“…Despite the availability of high-resolution structural data from X-ray crystallography 28 , 29 , mechanistic details of how SurA chaperones its OMP clients and delivers them to BAM, and how this is choreographed and controlled in the absence of ATP, have remained obscure 5 . Recent in vitro evidence has shed light on the mechanism of substrate interaction by SurA, and revealed that (1) the core domain of SurA contains the primary OMP binding sites 30 , 31 , (2) OMPs bind in a cradle formed between the core and P1 domains 30 , 31 , (3) the chaperone has a malleable architecture that responds to client binding 30 , 32 , 33 , and (4) clients bound to SurA can populate extended states 31 , 34 , 35 . However, despite this wealth of information, little is known about how SurA delivers its OMP clients to BAM to promote their folding.…”

“…How SurA and the other OMP folding factors work together to coordinate OMP assembly, and the precise mechanism by which BAM folds and inserts OMPs (that have a wide range of sizes and structural complexity 41 ) into the OM are still unclear 35 , 42 – 45 . In vitro and in silico evidence point to a role for BAM in membrane thinning and disordering of the OM bilayer to aid OMP folding 46 – 50 .…”

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

“…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 ). Heterologous expression and secretion of a lipase from Burkholderia in E. coli could be improved by 36% upon co-expression of Skp, but not SurA ( Narayanan et al, 2011 ).…”