Linkage between ATP Consumption and Mechanical Unfolding during the Protein Processing Reactions of an AAA+ Degradation Machine

Abstract

Proteolytic machines powered by ATP hydrolysis bind proteins with specific peptide tags, denature these substrates, and translocate them into a sequestered compartment for degradation. To determine how ATP is used during individual reaction steps, we assayed ClpXP degradation of ssrA-tagged titin variants with different stabilities in native and denatured forms. The rate of ATP turnover was 4-fold slower during denaturation than translocation. Importantly, this reduced turnover rate was constant during denaturation of native variants with different stabilities, but total ATP consumption increased with substrate stability, suggesting an iterative application of a uniform, mechanical unfolding force. Destabilization of substrate structure near the degradation tag accelerated degradation and dramatically reduced ATP consumption, revealing an important role for local protein stability in resisting denaturation. The ability to denature more stable proteins simply by using more ATP endows ClpX with a robust unfolding activity required for its biological roles in degradation and complex disassembly.

Geobacillus thermodenitrificans YjbH recognizes the C-terminal end of Bacillus subtilis Spx to accelerate Spx proteolysis by ClpXP

“…Our data support the hypothesis that GtYjbH binds the C terminus of Spx, altering Spx structure to expose a structural element for productive recognition by ClpX, and reducing non-productive binding of Spx with ClpX. Hence the weaker K m would result in faster degradation, since the unfolding of native protein is a ratelimiting step for protein degradation by ClpX (Kenniston et al, 2003). The strong interaction of GtYjbH and Spx could increase the effective concentration of Spx to the pore of ClpX, and then the equilibrium would shift towards the generation of degraded products (Davis et al, 2009).…”

Partitioning between unfolding and release of native domains during ClpXP degradation determines substrate selectivity and partial processing

“…Carboxymethylation of titin proteins was performed as reported in ref. 5, except for titin 10 , which required a 1,000-fold molar excess of iodoacetic acid (18). The structures of titin variants were assayed by CD spectroscopy, temperature melts, and tryptophan fluorescence (5).…”

“…We have been interested in understanding the coordination of substrate binding, denaturation, and translocation by the ClpXP protease of Escherichia coli (3)(4)(5)(6). Most substrates for this ATP-dependent protease have unstructured recognition sequences or degradation tags at their N or C terminus (7).…”

“…Studies of ssrA-tagged variants of the I27 domain of the muscle protein titin have been extremely useful in probing individual steps in the processing of protein substrates by ClpXP (5). The ssrA tag of the otherwise wild-type titin substrate is attached to a ␤-sheet with immense mechanical stability (16), and we have shown that proteolysis rates and ATP utilization during ClpXP degradation can vary dramatically when this ␤-sheet is destabilized by mutation (5).…”

“…The ssrA tag of the otherwise wild-type titin substrate is attached to a ␤-sheet with immense mechanical stability (16), and we have shown that proteolysis rates and ATP utilization during ClpXP degradation can vary dramatically when this ␤-sheet is destabilized by mutation (5). Importantly, chemical modification of cysteines in the hydrophobic core of titin results in global unfolding, and ClpXP degradation of carboxymethylated (CM) substrates can be used to assess the contribution of steps other than denaturation (5). During degradation of a single 121-residue molecule of ssrA-tagged titin, ClpXP hydrolyzes Ϸ100 molecules of ATP for translocation and uses an additional 500-600 ATPs for denaturation.…”