Coordinated gripping of substrate by subunits of a AAA+ proteolytic machine

Iosefson, Ohad; Nager, Andrew R.; Baker, Tania A.; Sauer, Robert T.

doi:10.1038/nchembio.1732

Cited by 60 publications

(102 citation statements)

References 36 publications

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“…Recent reports have focused on mechanistic aspects of the whole ClpXP unfolding machinery. [36][37][38] Beta-lactones, the only specific ClpP inhibitors reported to date, have been applied as tools to probe the binding pocket and mechanism of inhibition, as well as the transient stability of the ClpP tetradecamer and its conformational switching. 23,25,39 One additional consequence of beta-lactone-based ClpP inhibition is the reduction of virulence in pathogenic bacteria such as MRSA and the eradication of Mycobacteria that express two essential ClpP isoforms.…”

Section: Discussionmentioning

confidence: 99%

Phenyl Esters Are Potent Inhibitors of Caseinolytic Protease P and Reveal a Stereogenic Switch for Deoligomerization

et al. 2015

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Caseinolytic protease P (ClpP) represents a central bacterial degradation machinery that is involved in cell homeostasis and pathogenicity. The functional role of ClpP has been studied by genetic knockouts and through the use of beta-lactones, which remain the only specific inhibitors of ClpP discovered to date. Beta-lactones have served as chemical tools to manipulate ClpP in several organisms, however, their potency, selectivity and stability is limited. Despite detailed structural insights into the composition and conformational flexibility of the ClpP active site, no rational efforts to design specific non-beta-lactone inhibitors have been reported to date. In this work, an unbiased screen of more than 137,000 compounds was used to identify five phenyl ester compounds as highly potent ClpP inhibitors that were selective for bacterial, but not human ClpP.The potency of phenyl esters largely exceeded that of beta-lactones in ClpP peptidase and protease inhibition assays and displayed unique target selectivity in living S. aureus cells.Analytical studies revealed that while phenyl esters are cleaved like native peptide substrates, they remain covalently trapped as acyl-enzyme intermediates in the active site. The synthesis of 36 derivatives and subsequent structure-activity relationship (SAR) studies provided insights into conserved structural elements that are important for inhibition potency and acylation reactivity.Moreover, the stereochemistry of a methyl-substituent at the alpha position to the ester, resembling amino acid side chains in peptide substrates, impacted ClpP complex stability, causing either dissociation into heptamers or retention of the tetradecameric state. Mechanistic insights into this intriguing stereo switch and the phenyl ester binding mode were obtained by molecular docking experiments.

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Section: Discussionmentioning

confidence: 99%

Phenyl Esters Are Potent Inhibitors of Caseinolytic Protease P and Reveal a Stereogenic Switch for Deoligomerization

et al. 2015

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“…These loops feature the conserved hydrophobic [Tyr/Phe]-[Val/Leu/Ile]-Gly sequence pattern that has been previously suggested to drive substrate translocation in many ATP-dependent unfoldases such as HslU, ClpX, ClpA, LonA, FtsH, and PAN (37,38) (Fig. 4 C and E and SI Appendix, Figs.…”

Section: Resultsmentioning

confidence: 99%

Structural basis for dynamic regulation of the human 26S proteasome

Chen

et al. 2016

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

156

310

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The proteasome is the major engine of protein degradation in all eukaryotic cells. At the heart of this machine is a heterohexameric ring of AAA (ATPases associated with diverse cellular activities) proteins that unfolds ubiquitylated target proteins that are concurrently translocated into a proteolytic chamber and degraded into peptides. Using cryoelectron microscopy, we determined a near–atomic-resolution structure of the 2.5-MDa human proteasome in its ground state, as well as subnanometer-resolution structures of the holoenzyme in three alternative conformational states. The substrate-unfolding AAA-ATPase channel is narrowed by 10 inward-facing pore loops arranged into two helices that run in parallel with each other, one hydrophobic in character and the other highly charged. The gate of the core particle was unexpectedly found closed in the ground state and open in only one of the alternative states. Coordinated, stepwise conformational changes of the regulatory particle couple ATP hydrolysis to substrate translocation and regulate gating of the core particle, leading to processive degradation.

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“…As predicted, ClpXP and ClpAP degraded the N-tagged substrate much faster than the C-tagged substrate. For C-tagged substrates, three lowermolecular-weight bands corresponding to fragments with different numbers of titin I27 domains remaining were observed to accumulate during the degradation reaction (20,21), as expected if C-to-N unfolding/degradation of these domains by ClpXP or ClpAP is slow and enzyme dissociation often occurs, resulting in partially degraded products. By contrast, only one major intermediate or product accumulated during degradation of the N-tagged substrate, which corresponded to a fragment containing the C-terminal Halo domain.…”

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

confidence: 95%

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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Coordinated gripping of substrate by subunits of a AAA+ proteolytic machine

Cited by 60 publications

References 36 publications

Phenyl Esters Are Potent Inhibitors of Caseinolytic Protease P and Reveal a Stereogenic Switch for Deoligomerization

Phenyl Esters Are Potent Inhibitors of Caseinolytic Protease P and Reveal a Stereogenic Switch for Deoligomerization

Structural basis for dynamic regulation of the human 26S proteasome

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

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