Hinge–Linker Elements in the AAA+ Protein Unfoldase ClpX Mediate Intersubunit Communication, Assembly, and Mechanical Activity

Abstract

The ClpXP protease plays important roles in protein homeostasis and quality control. ClpX is a ringshaped AAA+ homohexamer that unfolds target proteins and translocates them into the ClpP peptidase for degradation. AAA+ modules in each ClpX subunitconsisting of a large AAA+ domain, a short hinge-linker, and a small AAA+ domainmediate the mechanical activities of the ring hexamer. Here, we investigate the roles of these hinge-linkers in ClpX function. Deleting one hinge-linker in a singlechain ClpX pseudohexamer dramatically decreases unfolding and degradation activity, in part by compromising formation of closed rings, protein-substrate binding, and ClpP binding. Covalently reclosing the broken hinge-linker interface rescues activity. Deleting one hinge-linker from a single-chain dimer or trimer prevents assembly of stable hexamers. Mutationally disrupting a hinge-linker preserves closed ring assembly but reduces ATP-hydrolysis cooperativity and degradation activity. These results indicate that hinge-linker length and flexibility are optimized for efficient substrate unfolding and support a model in which the hinge-linkers of ClpX facilitate efficient degradation both by maintaining proper ring geometry and facilitating subunit-subunit communication. This model informs our understanding of ClpX as well as the larger AAA+ family of motor proteins, which play diverse roles in converting chemical into mechanical energy in all cells.

Omnipresent Maxwell's demons orchestrate information management in living cells

“…Despite extensive study, complete understanding of how the nucleotide‐binding domains coordinate ATP binding and hydrolysis to polypeptide translocation is currently lacking. Again, the role of ClpX or ClpA has been interpreted as purely mechanical (Bell et al ., ), but we can be confident that, besides a likely mechanical action required to unfold their substrates, these proteins must also manage information, as previously discussed. A MxD activity via energy‐dependent selection of targets should be explored in priority.…”

Structures of the ATP-fueled ClpXP proteolytic machine bound to protein substrate

“…Experiments were performed in triplicate and reported values are averages ± SD. Degradation of CP7 GFP-ssrA (15 µM) by ClpX assayed as described (Bell et al, 2018). K M and V max were determined by fitting the average values of replicates to a hyperbolic equation.…”

“…Single-chain ClpX ∆N was used to ensure subunits of the pseudohexamer do not dissociate during sample preparation. This enzyme has been used previously for many biochemical and single-molecule studies (Martin et al, 2005;2008a;2008b;Aubin-Tam et al, 2011;Maillard et al, 2011;Glynn et al, 2012;Sen et al, 2013;Stinson et al, 2013;Cordova et al, 2014;Iosefson et al, 2105a;2015b;Olivares et al, 2017;Rodriguez-Aliaga et al, 2016;Amor et al, 2019;Bell et al, 2018). We purified enzymes separately and incubated ClpX ∆N (4 µM pseudohexamer), ClpP (2 µM 14-mer), and ATPγS (5 mM) for five min before vitrification.…”

“…The relative orientations of the large and small AAA+ domains were similar across all four structural classes for subunits at equivalent spiral positions, but conformational changes in the hinge resulted in different orientations or the large and small domains at many positions in the spiral for each hexamer ( Figure 5F). Changes in hinge length or deletion of one hinge largely eliminate ClpX function (Glynn et al, 2012;Bell et al, 2018). Thus, the conformational changes associated with a power stroke likely arise from changes in hinge conformations, whereas movements of different large-domain loops or the Nsubdomain mediate ring closure and asymmetric contacts with ClpP and substrate.…”

“…For RKH loop mutants with reduced substrate affinity, degradation was measured at high concentrations of fluorescent substrate, with excitation at an off-peak wavelength (excitation 420 nm; emission 511 nm). Degradation of fluorescein-labeled Arc-st11-ssrA by ClpX ∆N (0.3 µM hexamer) and ClpP 14 (0.9 µM) was assayed as described (Bell et al, 2018). K M and V max were determined by fitting the average values of replicates to a hyperbolic equation.…”