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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 uOMP aggregation, facilitate diffusion across the periplasm, and enhance OMP folding. FkpA primarily responds to heat-shock stress, but its mechanism is comparatively understudied. To determine FkpA chaperone function, we monitored the folding of a cognate client uOmpA171and found that FkpA increases the folded uOmpA171population but also slows the folding rate, dual functions distinct from the other periplasmic chaperones. The results indicate that FkpA behaves as a chaperone and not as a folding catalyst to directly influence the uOmpA171folding trajectory. We determine the binding affinity between FkpA and uOmpA171by globally fitting sedimentation velocity titrations and found it to be intermediate between the known affinities of Skp and SurA for uOMP clients. Notably, complex formation steeply depends on the urea concentration, suggestive of an extensive binding interface. Initial characterizations of the complex using photo-crosslinking indicates that the binding interface spans the inner surfaces of 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 uOMP folding and that it achieves this by utilizing an extensive chaperone-client interface.SignificanceThe periplasmic chaperone network is required for the survival and virulence of gram-negative bacteria. Here we find that the chaperone FkpA enhances outer membrane protein folding and tightly binds its clients with an extensive interaction interface. This modified holdase function of FkpA distinguishes it from other periplasmic chaperones and complements their functions to ensure robust outer membrane biogenesis.

Anfinsen (1961) demonstrated that the information for a protein to fold is entirely contained within its sequence. More recently, the importance of unfolded, but functional, proteins has been recognised. These so-called intrinsically disordered proteins (IDPs) lack a three-dimensional structure, but may fold upon interacting with a partner protein. Where is the folding information for these coupled folding and binding reactions contained? Is the amino acid sequence of the IDP the sole determinant of the folding reaction? Or are they dictated by the partner protein? We examine these questions by comparing and contrasting multiple interactions between IDPs and their structured partners, utilising the Bcl-2 family as a model system.