Dissection of Axial-Pore Loop Function during Unfolding and Translocation by a AAA+ Proteolytic Machine

Iosefson, Ohad; Olivares, Adrian O.; Baker, Tania A.; Sauer, Robert T.

doi:10.1016/j.celrep.2015.07.007

Cited by 49 publications

(79 citation statements)

References 30 publications

(73 reference statements)

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“…1D). This behavior is expected for fully cooperative unfolding and was previously observed for ClpXP unfolding of titin domains initiated at the C terminus (14,15,17). Following unfolding by ClpXP or ClpAP, translocation of the denatured domain resulted in a gradual reduction in bead-to-bead distance (Fig.…”

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

confidence: 64%

“…As shown here, single-molecule optical trapping experiments can answer these questions. Based on results reported here and elsewhere (14)(15)(16)(17)(27)(28)(29), both the ClpXP and ClpAP proteases unfold the titin I27 domain much faster and at far lower energetic costs when pulling from the N terminus as opposed to the C terminus. In principle, many factors could contribute to these differences.…”

Section: Discussionmentioning

confidence: 99%

“…For ensemble experiments, proteins successfully cross-linked to maleoyl-AH 6 AANDENYALAA-coo Single-Molecule Optical Trapping. Complexes of ClpXP or ClpAP and multidomain substrates were tethered between two beads trapped by 1,064-nm lasers in passive force-clamp mode as described (14)(15)(16)(17)35). Briefly, biotinylated, DNA-linked, multidomain substrates were attached to 1-μm streptavidin-coated polystyrene beads (Spherotech) that were loosely tethered to the surface of a glass coverslip via a DNA-linked glass-binding peptide aptamer.…”

Section: Methodsmentioning

confidence: 99%

“…We monitored single-molecule unfolding and translocation by ClpXP and ClpAP using a dual-laser optical trap in passive force-clamp mode as described (14)(15)(16)(17). ClpXP or ClpAP complexes containing biotinylated ClpP were immobilized to one streptavidin-coated bead (15).…”

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

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

Proc. Natl. Acad. Sci. U.S.A.

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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

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

confidence: 64%

Section: Discussionmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

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

confidence: 99%

See 2 more Smart Citations

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

Olivares

Kotamarthi

Stein

et al. 2017

Proc. Natl. Acad. Sci. U.S.A.

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“…The mechanical force for this process is generated from repeated cycles of ATP binding and hydrolysis. Substrates are pulled through the narrow channel of the unfoldase via attachment to pore loops located at the entrance and in the interior of the channel (65,(67)(68)(69)(70), which also play a role in substrate recognition in some AAA+ machines (5,(71)(72)(73). In the case of VAT, mutations of pore loop residues Y264 in D1 and W541/V542 in D2 cause severe defects on substrate unfolding and translocation (18).…”

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

Proc. Natl. Acad. Sci. U.S.A.

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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 ...

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New insights into structural and functional relationships between LonA proteases and ClpB chaperones

et al. 2019

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LonA proteases and ClpB chaperones are key components of the protein quality control system in bacterial cells. LonA proteases form a unique family of ATPases associated with diverse cellular activities (AAA + ) proteins due to the presence of an unusual N‐terminal region comprised of two domains: a β‐structured N domain and an α‐helical domain, including the coiled‐coil fragment, which is referred to as HI(CC). The arrangement of helices in the HI(CC) domain is reminiscent of the structure of the H1 domain of the first AAA + module of ClpB chaperones. It has been hypothesized that LonA proteases with a single AAA + module may also contain a part of another AAA + module, the full version of which is present in ClpB. Here, we established and tested the structural basis of this hypothesis using the known crystal structures of various fragments of LonA proteases and ClpB chaperones, as well as the newly determined structure of the Escherichia coli LonA fragment (235–584). The similarities and differences in the corresponding domains of LonA proteases and ClpB chaperones were examined in structural terms. The results of our analysis, complemented by the finding of a singular match in the location of the most conserved axial pore‐1 loop between the LonA NB domain and the NB2 domain of ClpB, support our hypothesis that there is a structural and functional relationship between two coiled–coil fragments and implies a similar mechanism of engagement of the pore‐1 loops in the AAA + modules of LonAs and ClpBs.

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Dissection of Axial-Pore Loop Function during Unfolding and Translocation by a AAA+ Proteolytic Machine

Cited by 49 publications

References 30 publications

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

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

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

New insights into structural and functional relationships between LonA proteases and ClpB chaperones

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