SurA, Skp, FkpA, and DegP constitute a chaperone network that ensures biogenesis of outer membrane proteins (OMPs) in Gram‐negative bacteria. Both Skp and FkpA are holdases that prevent the self‐aggregation of unfolded OMPs, whereas SurA accelerates folding and DegP is a protease. None of these chaperones is essential, and we address here how functional plasticity is manifested in nine known null strains. Using a comprehensive computational model of this network termed OMPBioM, our results suggest that a threshold level of steady state holdase occupancy by chaperones is required, but the cell is agnostic to the specific holdase molecule fulfilling this function. In addition to its foldase activity, SurA moonlights as a holdase when there is no expression of Skp and FkpA. We further interrogate the importance of chaperone–client complex lifetime by conducting simulations using lifetime values for Skp complexes that range in length by six orders of magnitude. This analysis suggests that transient occupancy of durations much shorter than the
Outer membrane protein (OMP) biogenesis in gram‐negative bacteria is managed by a network of periplasmic chaperones that includes SurA, Skp, and FkpA. These chaperones bind unfolded OMPs (uOMPs) in dynamic conformational ensembles to suppress aggregation, facilitate diffusion across the periplasm, and enhance folding. FkpA primarily responds to heat‐shock stress, but its mechanism is comparatively understudied. To determine FkpA chaperone function in the context of OMP folding, we monitored the folding of three OMPs and found that FkpA, unlike other periplasmic chaperones, increases the folded yield but decreases the folding rate of OMPs. The results indicate that FkpA behaves as a chaperone and not as a folding catalyst to influence the OMP folding trajectory. Consistent with the folding assay results, FkpA binds all three uOMPs as determined by sedimentation velocity (SV) and photo‐crosslinking experiments. We determine the binding affinity between FkpA and uOmpA171by globally fitting SV titrations and find it to be intermediate between the known affinities of Skp and SurA for uOMP clients. Notably, complex formation steeply depends on the urea concentration, suggesting an extensive binding interface. Initial characterizations of the complex using photo‐crosslinking indicate that the binding interface spans the entire FkpA molecule. In contrast to prior findings, folding and binding experiments performed using subdomain constructs of FkpA demonstrate that the full‐length chaperone is required for full activity. Together these results support that FkpA has a distinct and direct effect on OMP folding that it achieves by utilizing an extensive chaperone‐client interface to tightly bind clients.
more » « less- Award ID(s):
- 1931211
- NSF-PAR ID:
- 10419849
- Publisher / Repository:
- Wiley Blackwell (John Wiley & Sons)
- Date Published:
- Journal Name:
- Protein Science
- Volume:
- 32
- Issue:
- 4
- ISSN:
- 0961-8368
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
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