Flexible client-dependent cages in the assembly landscape of the periplasmic protease-chaperone DegP

Abstract

The periplasmic protein DegP, that is implicated in virulence factor transport leading to pathogenicity, is a bi-functional protease and chaperone that maintains protein homeostasis in gram-negative bacteria. To perform these functions, DegP captures clients inside cage-like structures, which we have recently shown to form through the reorganization of high-order preformed apo-oligomers, consisting of trimeric building blocks, that are structurally distinct from client-bound cages. Our previous studies suggested that these apo oligomers may allow DegP to encapsulate clients of various sizes under protein folding stresses by forming cage ensembles that can include extremely large cage particles. To explore the relation between cage and substrate sizes, we engineered a series of DegP clients of increasing hydrodynamic radii and analyzed their influence on DegP cage formation. We used dynamic light scattering and cryogenic electron microscopy to characterize the hydrodynamic properties and structures of the DegP cages that are adopted in response to each client. We present a series of flexible cage structures including novel 30mer and 60mer particles. Key interactions between DegP trimers and the bound clients that stabilize the cage assemblies and prime the clients for catalysis are revealed. We also provide evidence that DegP can form cages which approach subcellular organelles in terms of size. Significance statementGram-negative pathogens export virulence factors that interfere with the function of host cells. This process is mediated by DegP, a protein which controls protein homeostasis in the periplasm of these bacteria and thus is a target for the development of novel antibiotics. DegP operates by incorporating client proteins inside cage-like structures to either recycle them or protect them from aggregation. Using a combination of dynamic light scattering measurements and cryogenic electron microscopy, we have shown that DegP can adopt many types of cages, some as large as subcellular organelles, depending on the size of the engaged client. This property likely enables DegP to capture different sized clients in response to protein misfolding stresses.