Tania A. Baker

Proteomic discovery of cellular substrates of the ClpXP protease reveals five classes of ClpX-recognition signals.

ClpXP is a protease involved in DNA damage repair, stationary-phase gene expression, and ssrA-mediated protein quality control. To date, however, only a handful of ClpXP substrates have been identified. Using a tagged and inactive variant of ClpP, substrates of E. coli ClpXP were trapped in vivo, purified, and identified by mass spectrometry. The more than 50 trapped proteins include transcription factors, metabolic enzymes, and proteins involved in the starvation and oxidative stress responses. Analysis of the sequences of the trapped proteins revealed five recurring motifs: two located at the C terminus of proteins, and three N-terminal motifs. Deletion analysis, fusion proteins, and point mutations established that sequences from each motif class targeted proteins for degradation by ClpXP. These results represent a description of general rules governing substrate recognition by a AAA+ family ATPase and suggest strategies for regulation of protein degradation.

AAA+ Proteases: ATP-Fueled Machines of Protein Destruction

AAA+ family proteolytic machines (ClpXP, ClpAP, ClpCP, HslUV, Lon, FtsH, PAN/20S, and the 26S proteasome) perform protein quality control and are used in regulatory circuits in all cells. These machines contain a compartmental protease, with active sites sequestered in an interior chamber, and a hexameric ring of AAA+ ATPases. Substrate proteins are tethered to the ring, either directly or via adaptor proteins. An unstructured region of the substrate is engaged in the axial pore of the AAA+ ring, and cycles of ATP binding/hydrolysis drive conformational changes that create pulses of pulling that denature the substrate and translocate the unfolded polypeptide through the pore and into the degradation chamber. Here, we review our current understanding of the molecular mechanisms of substrate recognition, adaptor function, and ATP-fueled unfolding and translocation. The unfolding activities of these and related AAA+ machines can also be used to disassemble or remodel macromolecular complexes and to resolubilize aggregates.

Sculpting the Proteome with AAA+ Proteases and Disassembly Machines

Machines of protein destruction-including energy-dependent proteases and disassembly chaperones of the AAA(+) ATPase family-function in all kingdoms of life to sculpt the cellular proteome, ensuring that unnecessary and dangerous proteins are eliminated and biological responses to environmental change are rapidly and properly regulated. Exciting progress has been made in understanding how AAA(+) machines recognize specific proteins as targets and then carry out ATP-dependent dismantling of the tertiary and/or quaternary structure of these molecules during the processes of protein degradation and the disassembly of macromolecular complexes.

Rebuilt AAA + motors reveal operating principles for ATP-fuelled machines.

Hexameric ring-shaped ATPases of the AAA + (for ATPases associated with various cellular activities) superfamily power cellular processes in which macromolecular structures and complexes are dismantled or denatured, but the mechanisms used by these machine-like enzymes are poorly understood. By covalently linking active and inactive subunits of the ATPase ClpX to form hexamers, here we show that diverse geometric arrangements can support the enzymatic unfolding of protein substrates and translocation of the denatured polypeptide into the ClpP peptidase for degradation. These studies indicate that the ClpX power stroke is generated by ATP hydrolysis in a single subunit, rule out concerted and strict sequential ATP hydrolysis models, and provide evidence for a probabilistic sequence of nucleotide hydrolysis. This mechanism would allow any ClpX subunit in contact with a translocating polypeptide to hydrolyse ATP to drive substrate spooling into ClpP, and would prevent stalling if one subunit failed to bind or hydrolyse ATP. Energy-dependent machines with highly diverse quaternary architectures and molecular functions could operate by similar asymmetric mechanisms.

Polymerases and the Replisome: Machines within Machines

We wish to thank Bruce Stillman, Jacqueline Lees, Alan Grossman, Ilana Goldhaber-Gordon, Glenn Sanders, Yong-In Kim, and Oscar Aparicio for comments on the manuscript. Work in S. P. B.s laboratory is supported by an NIH grant (GM-52339), the Searle Foundation, and the Rita Allen Foundation. Work in T. A. B.s laboratory is supported by an NIH grant (GM-499224) and by the Howard Hughes Medical Institute. T. A. B. is an employee of the Howard Hughes Medical Institute and a recipient of a National Science Foundation Young Investigator Award.

Dynamics of Substrate Denaturation and Translocation by the ClpXP Degradation Machine

ClpXP is a protein machine composed of the ClpX ATPase, a member of the Clp/Hsp100 family of remodeling enzymes, and the ClpP peptidase. Here, ClpX and ClpXP are shown to catalyze denaturation of GFP modified with an ssrA degradation tag. ClpX translocates this denatured protein into the proteolytic chamber of ClpP and, when proteolysis is blocked, also catalyzes release of denatured GFP-ssrA from ClpP in a reaction that requires ATP and additional substrate. Kinetic experiments reveal that multiple reaction steps require collaboration between ClpX and ClpP and that denaturation is the rate-determining step in degradation. These insights into the mechanism of ClpXP explain how it executes efficient degradation in a manner that is highly specific for tagged proteins, irrespective of their intrinsic stabilities.

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

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.

Overlapping recognition determinants within the ssrA degradation tag allow modulation of proteolysis

The ssrA tag, an 11-aa peptide added to the C terminus of proteins stalled during translation, targets proteins for degradation by ClpXP and ClpAP. Mutational analysis of the ssrA tag reveals independent, but overlapping determinants for its interactions with ClpX, ClpA, and SspB, a specificity-enhancing factor for ClpX. ClpX interacts with residues 9–11 at the C terminus of the tag, whereas ClpA recognizes positions 8–10 in addition to residues 1–2 at the N terminus. SspB interacts with residues 1–4 and 7, N-terminal to the ClpX-binding determinants, but overlapping the ClpA determinants. As a result, SspB and ClpX work together to recognize ssrA-tagged substrates efficiently, whereas SspB inhibits recognition of these substrates by ClpA. Thus, dissection of the recognition signals within the ssrA tag provides insight into how multiple proteins function in concert to modulate proteolysis.

Structures of asymmetric ClpX hexamers reveal nucleotide-dependent motions in a AAA+ protein-unfolding machine.

ClpX is a AAA+ machine that uses the energy of ATP binding and hydrolysis to unfold native proteins and translocate unfolded polypeptides into the ClpP peptidase. The crystal structures presented here reveal striking asymmetry in ring hexamers of nucleotide-free and nucleotide-bound ClpX. Asymmetry arises from large changes in rotation between the large and small AAA+ domains of individual subunits. These differences prevent nucleotide binding to two subunits, generate a staggered arrangement of ClpX subunits and pore loops around the hexameric ring, and provide a mechanism for coupling conformational changes caused by ATP binding or hydrolysis in one subunit to flexing motions of the entire ring. Our structures explain numerous solution studies of ClpX function, predict mechanisms for pore elasticity during translocation of irregular polypeptides, and suggest how repetitive conformational changes might be coupled to mechanical work during the ATPase cycle of ClpX and related molecular machines.

Pore loops of the AAA+ ClpX machine grip substrates to drive translocation and unfolding

Proteolytic AAA+ unfoldases use ATP hydrolysis to power conformational changes that mechanically denature protein substrates and then translocate the polypeptide through a narrow pore into a degradation chamber. We show that a tyrosine residue in a pore loop of the hexameric ClpX unfoldase links ATP hydrolysis to mechanical work by gripping substrates during unfolding and translocation. Removal of the aromatic ring in even a few ClpX subunits results in slippage, frequent failure to denature the substrate and an enormous increase in the energetic cost of substrate unfolding. The tyrosine residue is part of a conserved aromatic-hydrophobic motif, and the effects of mutations in both residues vary with the nucleotide state of the resident subunit. These results support a model in which nucleotide-dependent conformational changes in these pore loops drive substrate translocation and unfolding, with the aromatic ring transmitting force to the polypeptide substrate.