Stochastic but Highly Coordinated Protein Unfolding and Translocation by the ClpXP Proteolytic Machine

AAA+ proteases and remodeling machines couple hydrolysis of ATP to mechanical unfolding and translocation of proteins following recognition of sequence tags called degrons. Here, we use singlemolecule optical trapping to determine the mechanochemistry of two AAA+ proteases, Escherichia coli ClpXP and ClpAP, as they unfold and translocate substrates containing multiple copies of the titin I27 domain during degradation initiated from the N terminus. Previous studies characterized degradation of related substrates with C-terminal degrons. We find that ClpXP and ClpAP unfold the wild-type titin I27 domain and a destabilized variant far more rapidly when pulling from the N terminus, whereas translocation speed is reduced only modestly in the N-to-C direction. These measurements establish the role of directionality in mechanical protein degradation, show that degron placement can change whether unfolding or translocation is rate limiting, and establish that one or a few power strokes are sufficient to unfold some protein domains.protein degradation | AAA+ proteases | directional unfolding | AAA+ motors

Section: Single-molecule Degradation Of a Substrate Bearing An N-termmentioning

confidence: 66%

Section: Single-molecule Degradation Of a Substrate Bearing An N-termmentioning

confidence: 99%

mentioning

confidence: 99%

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Effect of directional pulling on mechanical protein degradation by ATP-dependent proteolytic machines

Olivares

Kotamarthi

Stein

et al. 2017

“…This type of helical arrangement is often observed for other AAA+ proteins, such as the yeast replication factor C (61), the bacterial DnaA AAA+ domain (62), the bacterial IstB domain (63), and the eukaryotic MCM E1 helicase (64), implying a potential evolutionary conservation in the architecture of these proteins despite varying structural features and distinct biological functions. It has been proposed that VAT functions via a substrate threading mechanism (18) similar to the mechanism used by other AAA+ unfoldases, such as ClpX and ClpA (65,66), whereby protein substrates are unfolded and threaded through the central pore of the unfoldase and subsequently passed into the degradation chamber of the 20S proteasome CP. The mechanical force for this process is generated from repeated cycles of ATP binding and hydrolysis.…”

Section: Discussionmentioning

confidence: 99%

Unfolding the mechanism of the AAA+ unfoldase VAT by a combined cryo-EM, solution NMR study

Huang

Ripstein

Augustyniak

et al. 2016

The AAA+ (ATPases associated with a variety of cellular activities) enzymes play critical roles in a variety of homeostatic processes in all kingdoms of life. Valosin-containing protein-like ATPase of Thermoplasma acidophilum (VAT), the archaeal homolog of the ubiquitous AAA+ protein Cdc48/p97, functions in concert with the 20S proteasome by unfolding substrates and passing them on for degradation. Here, we present electron cryomicroscopy (cryo-EM) maps showing that VAT undergoes large conformational rearrangements during its ATP hydrolysis cycle that differ dramatically from the conformational states observed for Cdc48/p97. We validate key features of the model with biochemical and solution methyl-transverse relaxation optimized spectroscopY (TROSY) NMR experiments and suggest a mechanism for coupling the energy of nucleotide hydrolysis to substrate unfolding. These findings illustrate the unique complementarity between cryo-EM and solution NMR for studies of molecular machines, showing that the structural properties of VAT, as well as the population distributions of conformers, are similar in the frozen specimens used for cryo-EM and in the solution phase where NMR spectra are recorded.T he AAA+ enzymes (ATPases associated with a variety of cellular activities) use the energy of ATP hydrolysis to carry out a myriad of different biological functions that are critical to cellular homeostasis. These molecular machines are typically barrel-shaped, containing a narrow channel that serves to traffic substrates through the enzyme (1-4). In the case of the heat shock protein 104 (Hsp104) AAA+ class of chaperone, for example, substrates are dissociated from aggregates via a pulling motion that forces individual polypeptide chains through the central pore of the enzyme (1, 5). The unfolded proteins that emerge subsequently refold spontaneously or are acted on by additional chaperones that aid in their refolding (6). Other AAA+ enzymes act synergistically with proteases, unfolding targeted substrates and passing them directly to the proteases, where they are subsequently degraded (7). In this way, proteins that have become damaged or that are no longer required can be removed before their accumulation disrupts cellular function (8). A variety of different AAA+ unfoldases have been characterized, including Rpt1-6 in the 19S proteasome regulatory particle in eukaryotes (9-11), PAN in archaea (12-14), Mpa/ARC in Actinobacteria (15), ClpA/X (2) and HslU (3) in bacteria, and VAT in the Thermoplasma acidophilum archaebacterium (16)(17)(18)(19)(20).VAT is an ∼500-kDa homohexamer, with each monomer containing two tandem AAA+ domains, referred to as D1 and D2, and an N-terminal domain (NTD) distinct from the NTD of other AAA+ enzymes (16) (Fig. 1A). Both D1 and D2 are homologous to the single AAA+ modules of PAN and Rpt1-6 (1, 16). Biochemical studies have shown that VAT unfolds globular proteins and interacts directly with the 20S core particle (CP) of the proteasome, leading to enhanced proteolytic activity for both folded and ...

“…For the F1 ATPase, it has been established that ATPase domains hydrolyze ATP continuously in a strictly consecutive manner around the ring (2). In contrast, in another single-ring ATPase, ClpX, several ATPase domains hydrolyze ATP in sporadic bursts, either simultaneously or in short succession (3,(8)(9)(10). For double-ring ATPases, the coordination between ATPase subunits is even more complex, as there may be communication both within a given ring and between the two rings.…”

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confidence: 99%

Unique double-ring structure of the peroxisomal Pex1/Pex6 ATPase complex revealed by cryo-electron microscopy

Blok

Tan

Wang

et al. 2015

Members of the AAA family of ATPases assemble into hexameric double rings and perform vital functions, yet their molecular mechanisms remain poorly understood. Here, we report structures of the Pex1/Pex6 complex; mutations in these proteins frequently cause peroxisomal diseases. The structures were determined in the presence of different nucleotides by cryo-electron microscopy. Models were generated using a computational approach that combines Monte Carlo placement of structurally homologous domains into density maps with energy minimization and refinement protocols. Pex1 and Pex6 alternate in an unprecedented hexameric double ring. Each protein has two N-terminal domains, N1 and N2, structurally related to the single N domains in p97 and N-ethylmaleimide sensitive factor (NSF); N1 of Pex1 is mobile, but the others are packed against the double ring. The N-terminal ATPase domains are inactive, forming a symmetric D1 ring, whereas the C-terminal domains are active, likely in different nucleotide states, and form an asymmetric D2 ring. These results suggest how subunit activity is coordinated and indicate striking similarities between Pex1/Pex6 and p97, supporting the hypothesis that the Pex1/Pex6 complex has a role in peroxisomal protein import analogous to p97 in ER-associated protein degradation.Pex1 | Pex6 | AAA ATPase | cryo-electron microscopy | peroxisome T he AAA (ATPases associated with diverse cellular activities) family of ATPases contains a large number of proteins that perform important functions in cells (1). Many members of this family form hexamers. These hexamers consist either of a single ring of ATPase domains or of stacked rings formed by tandem ATPase domains in a single polypeptide chain (type I and II ATPases, respectively). Both classes of AAA ATPases often use the energy of ATP hydrolysis to move macromolecules. Examples of single-ring ATPases include the F1 ATPase (2), which rotates a polypeptide inside its central pore, ClpX (3), which moves polypeptides into the proteolytic chamber of ClpP, and the helicase gp4 of T7 phage (4), which pulls a DNA strand through its center. Double-ring ATPases generally act on polypeptides. Prominent members of this family include N-ethylmaleimide sensitive factor (NSF), p97/VCP (valosin-containing protein), and Hsp104 in eukaryotes and ClpB in bacteria (5-7). NSF dissociates SNARE complexes generated during membrane fusion, p97/VCP plays a role in many processes, including ERassociated protein degradation (ERAD), and Hsp104 and ClpB disassemble protein aggregates.The molecular mechanism of many hexameric AAA ATPases is only poorly understood, particularly for those that move polypeptide chains. In addition, the mechanism appears to differ among the known examples. For the F1 ATPase, it has been established that ATPase domains hydrolyze ATP continuously in a strictly consecutive manner around the ring (2). In contrast, in another single-ring ATPase, ClpX, several ATPase domains hydrolyze ATP in sporadic bursts, either simultaneously or in short succession...