Mechanical force is used to drive vectorial work in many cellular processes. In organisms from bacteria to humans, molecular machines of the AAA+ family (A
ssociated with various cellular A
ctivities) couple energy derived from ATP binding and hydrolysis to the degradation, remodeling, or movement of macromolecules1,2
. These enzymes work as protein unfoldases, disaggregating machines, DNA and RNA helicases, macromolecular pumps, and microtubule motor and severing proteins. How these machines function is an active area of investigation.
ClpX is a hexameric AAA+ machine that unfolds protein substrates and translocates them into the degradation chamber of ClpP, a self-compartmentalized peptidase3
(). Crystal structures of ClpX have been solved in a open helical conformation and as a ring hexamer4,5
. Lock-washer and helical conformations of some AAA+ machines are thought to be the relevant species for biological function6–10
. For ClpX, present evidence supports a functional role for the ring conformation, but it is unclear if the ring opens during the conformational fluctuations that power protein unfolding or when multiple polypeptides need to be translocated during the degradation of disulfide-bonded substrates3
. Another aspect of understanding ClpX function is determining which structural elements provide the flexibility to adopt the different conformations needed for machine function, while maintaining a sufficiently rigid overall structure to apply the forces needed to unfold native proteins. Macroscopic machines typically consist of rigid components that are flexibly linked to allow them to move with respect to each other. For example, the pistons of an internal combustion engine connect to the crankshaft in a way that allows piston movement to rotate the crankshaft, which can then be coupled to many different kinds of mechanical work.
Figure 1 The ClpX6 ring is stabilized by rigid-body packing between subunits. (a) In the ATP-dependent ClpXP protease, a ClpX hexamer recognizes and unfolds protein substrates, and then translocates them into the degradation chamber of ClpP14 for proteolysis. (more ...)
The AAA+ module of ClpX consists of a large domain (residues 65–314), a short hinge (residues 315–318), and a small domain (residues 319–424)4,11–12
. In ring hexamers, the small AAA+ domains are on the periphery of the ring, whereas the large AAA+ domains surround an axial pore, which is lined by loops that help the enzyme grip and translocate substrates5,13–17
(). Although ClpX subunits are identical in sequence, they adopt distinct conformations and roles during the ATPase cycle5,15–16,18
. In four loadable subunits, a cleft between the large and small AAA+ domains provides a binding site for ATP or ADP, with nucleotide contacts made by each domain and by the intervening hinge (); these subunits display a range of nucleotide-binding properties and modest changes in the nucleotide-dependent conformation of the hinge5,18
. Two opposed unloadable subunits in the ClpX ring do not bind nucleotide, because a radically different hinge conformation rotates the large and small AAA+ domains enough to destroy the binding cleft. Despite inherent asymmetry of the ring, each large AAA+ domain packs against the small AAA+ domain of the neighboring subunit in a nearly invariant manner5
(). A static ring can therefore be viewed as six rigid-body units, each spanning two subunits, connected by six hinges ().
Here, we ask if a ClpX hexamer functions as a closed ring and if the rigid-body packing between subunits is preserved throughout all of the different conformational changes that define the ATP-fueled mechanical cycle. Specifically, we engineer and assay the functional effects of covalent ties that tightly restrict the rigid-body packing between neighboring large and small domains in hexamers. Strikingly, mutants constrained in this fashion have a topologically closed ring but mediate robust unfolding and degradation. These results in combination with the properties of a hinge mutant strongly support a model in which conformational changes, which originate in one hinge and its flanking domains as a consequence of ATP binding or hydrolysis, are propagated around the AAA+ ring via the topologically constrained set of rigid-body units and hinges, producing coupled ring motions that power substrate unfolding.