Regulatory coiled-coil domains promote head-to-head assemblies of AAA+ chaperones essential for tunable activity control

Abstract

Ring-forming AAA+ chaperones exert ATP-fueled substrate unfolding by threading through a central pore. This activity is potentially harmful requiring mechanisms for tight repression and substrate-specific activation. The AAA+ chaperone ClpC with the peptidase ClpP forms a bacterial protease essential to virulence and stress resistance. The adaptor MecA activates ClpC by targeting substrates and stimulating ClpC ATPase activity. We show how ClpC is repressed in its ground state by determining ClpC cryo-EM structures with and without MecA. ClpC forms large two-helical assemblies that associate via head-to-head contacts between coiled-coil middle domains (MDs). MecA converts this resting state to an active planar ring structure by binding to MD interaction sites. Loss of ClpC repression in MD mutants causes constitutive activation and severe cellular toxicity. These findings unravel an unexpected regulatory concept executed by coiled-coil MDs to tightly control AAA+ chaperone activity.

The antibiotic cyclomarin blocks arginine-phosphate–induced millisecond dynamics in the N-terminal domain of ClpC1 from Mycobacterium tuberculosis

“…This difference could derive from a concentrationdependent oligomeric equilibrium, which would explain why MtbClpC1 seems larger in our study as compared with the SEC-MALS data presented by Carroni et al (27). In fact, crosslinking data from the same study already suggests the presence of complexes bigger than the EM decameric structure (27). Although we could not detect the hexamer in solution, the fact that under the same conditions ClpC1 is active, catalyzing the degradation of GFP-ssrA and casein in association with ClpP1P2, suggests that a part of the population exists as a hexamer.…”

“…Considering the similarities between our data and the previously reported cryo-EM structure, it is likely that MtbClpC1 forms a resting state, with a large part of the population representing even higher oligomers than decamers. This difference could derive from a concentrationdependent oligomeric equilibrium, which would explain why MtbClpC1 seems larger in our study as compared with the SEC-MALS data presented by Carroni et al (27). In fact, crosslinking data from the same study already suggests the presence of complexes bigger than the EM decameric structure (27).…”

“…Adding further complexity to the study of the system, we show here that MtbClpC1 can exist in an equilibrium between different oligomeric states. The existence of a resting decameric state formed by the association of head-to-head contacts between coiled-coil middle domains of ClpC was recently described by Carroni et al (27). The middle domains were proposed to repress the activity of ClpC by forming a highly dynamic resting state that blocks substrate binding or ClpP interaction.…”

“…Recently, Carroni et al (27), using cryo-electron microscopy (cryo-EM) and mutagenesis, showed that Staphylococcus aureus ClpC (SaClpC) exists in a decameric resting state formed through ClpC middle domains establishing intermolecular head-to-head contacts. This head-to-head contact allows the docking of two layers of ClpC molecules arranged in a helical conformation.…”

A stutter in the coiled coil domain ofE.colico-chaperone GrpE connects structure with function

“…Coiled-coil domains are common structural motifs present in various globular proteins and show diverse functions like assisting in proteins oligomerization, providing mechanical and thermal stability, and facilitating protein-protein interactions 22,23 . Coiled coils are also found in molecular chaperones, where they regulate protein-protein interactions and ensure proper protein homeostasis 24,25 . Some coiledcoils in bacteria have been reported to play a significant role in virulence and host-pathogen interactions 26,27 .…”