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.

Exploring Host–Guest Interactions within a 600 kDa DegP Protease Cage Complex Using Hydrodynamics Measurements and Methyl-TROSY NMR

“…The redistribution of the apo DegP ensemble could be described in terms of two pathways (Figure 1A, middle), one of which serves to buffer the concentration of free trimers through the formation of M 6A (path A, blue) and is predominantly operative at lower temperatures, while the other (path B, red) becomes active at higher temperatures and features the assembly of cage-like species mediated by relatively weakly associating and rapidly exchanging trimer units. Our studies 18,31 have revealed that M 6A , corresponding to the closed hexamer that was initially characterized via X-ray crystallography, 10 is not appreciably populated under conditions where bacteria grow efficiently (e.g., human host temperatures and salt concentrations) and therefore does not appear to play a major role in modulating the availability of trimers for engaging clients in these circumstances. Rather, the network of preorganized assemblies in path B underlies the formation of discrete cage complexes and shifts toward much larger species under stress conditions (e.g., heat shock and the associated overexpression of DegP 21 ) to quickly capture clients.…”

“…9 In recent investigations from our group, we have explored the energetics of DegP's self-assembly landscape in the absence and presence of model substrates under a variety of simulated physiological conditions so as to understand how cage remodeling is involved in the maintenance of protein homeostasis. 18,31 We demonstrated that in the absence of client proteins, the energy landscape of DegP rapidly changes in response to stress. The redistribution of the apo DegP ensemble could be described in terms of two pathways (Figure 1A, middle), one of which serves to buffer the concentration of free trimers through the formation of M 6A (path A, blue) and is predominantly operative at lower temperatures, while the other (path B, red) becomes active at higher temperatures and features the assembly of cage-like species mediated by relatively weakly associating and rapidly exchanging trimer units.…”

“…Samples for DLS measurements were prepared as described previously. 18,31 Autocorrelation functions for each sample well in the plate reader format instrument were measured over the temperature range 5−50 °C in discrete increments of 2.5 °C for a total sampling of 19 temperature points. Each well was measured 25 times per temperature with an acquisition time of 1 s per measurement.…”

“…18 Addition of increasing molar equivalents of hTRF1 resulted in the buildup of a dominant ∼17S species, consistent with an hTRF1-bound 12mer cage structure (Figure 1C,D, dark blue curves), which is the predominant species formed through interactions with this client. 18,31 The exact position of the 12mer varies between the two S210A DegP profiles ( The inset shows a superposition of 13 C-1 H HMQC spectra recorded for a concentration series of apo ILVM S210A DegP (0.025−2 mM protomer concentration, dark red to dark blue contours). 18 The pink star highlights the L276δ1 chemical shift obtained from a trimer mutant of DegP, i.e., the limit where no intertrimer interactions are present, and the white star is placed at the chemical shift position of the L276δ1 methyl group in the context of the 12mer cage where all PDZ1 i :PDZ2 j contacts are adopted (same white star is shown in each of the main panels).…”

“…18 Using a panel of engineered client proteins, we further showed that substrate-bound DegP forms distributions of expandable cage particles whose dimensions depend on the size of the substrate within the cage interiors. 31 Remarkably, these cage ensembles can include species with sizes up to subcellular organelles. Together, the rapid assembly and extraordinary structural plasticity of the DegP trimers enables the engagement of different types of clients in the periplasm.…”