Substrate processing by the Cdc48 ATPase complex is initiated by ubiquitin unfolding

Twomey, Edward C.; Ji, Zhejian; Wales, Thomas E.; Bodnar, Nicholas O.; Ficarro, Scott B.; Marto, Jarrod A.; Engen, John R.; Rapoport, Tom A.

doi:10.1126/science.aax1033

Cited by 258 publications

(441 citation statements)

References 64 publications

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“…4a). Recently, the cryo-EM structure of yeast Cdc48 in complex with Npl4/Ufd1 and K48-linked polyubiquitinated Eos showed that an unfolded ubiquitin moiety binds in the groove of Npl4 and extends all the way into the pore of Cdc48 in the 7 absence of ATP hydrolysis 5 . By contrast, none of the states showed any density in the groove of Npl4 ( Fig.…”

Section: Seesaw Motion Of Npl4 Is Important For the Unfolding Activitmentioning

confidence: 99%

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Seesaw Conformations of Npl4 in the Human p97 Complex and the Inhibitory Mechanism of a Disulfiram Derivative

Pan

Zheng

et al. 2020

Preprint

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p97, also known as valosin-containing protein (VCP) or Cdc48, plays a central role in cellular protein homeostasis 1 . Human p97 mutations are associated with several neurodegenerative diseases 2,3 . Targeting p97 and its cofactors is a strategy for cancer drug development 4 . Despite significant structural insights into the fungal homolog Cdc48 5-7 , little is known about how human p97 interacts with its cofactors. Recently, the anti-alcohol abuse drug disulfiram was found to target cancer through Npl4, a cofactor of p97 8 , but the molecular mechanism remains elusive. Here, using single-particle cryo-electron microscopy (cryo-EM), we uncovered three Npl4 conformational states in complex with human p97 before ATP hydrolysis.The motion of Npl4 results from its zinc finger motifs interacting with the N domain of p97, which is essential for the unfolding activity of p97. In vitro and cell-based assays showed that under oxidative conditions, the disulfiram derivative bis-(diethyldithiocarbamate)-copper (CuET) inhibits p97 function by releasing cupric ions, which disrupt the zinc finger motifs of Npl4, locking the essential conformational switch of the complex.Recently, the cryo-electron microscopy (cryo-EM) structures of the thermophilic fungus Cdc48-Npl4/Ufd1 complex 6 and the yeast Cdc48-Npl4/Ufd1-Eos complex 5 were reported. In

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Section: Seesaw Motion Of Npl4 Is Important For the Unfolding Activitmentioning

confidence: 99%

“…Furthermore, we revealed that the disulfiram derivative bis-(diethyldithiocarbamate)-copper (CuET) inhibits the unfolding activity of p97 and locks the conformational changes between p97 and Npl4/Ufd1 by releasing cupric ions under oxidative conditions. 5…”

Section: Introductionmentioning

confidence: 99%

Seesaw Conformations of Npl4 in the Human p97 Complex and the Inhibitory Mechanism of a Disulfiram Derivative

Pan

Zheng

et al. 2020

Preprint

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“…Our structural analysis reveals that TgMcrB AAA forms an asymmetric hexamer, similar to the architecture adopted by many other AAA+ family proteins (de la Pena et al, 2018; Enemark and Joshua-Tor, 2006; Gates et al, 2017; Puchades et al, 2017; Ripstein et al, 2017; Twomey et al, 2019; Zehr et al, 2017; Zhao et al, 2015). Asymmetry appears to be maintained by the conformation of key interface residues – Arg360, Glu527 and Tyr530 in one monomer and Arg414, Asp420 and Arg424 in its neighbor – acting in trans .…”

Section: Discussionmentioning

confidence: 52%

Structural asymmetry governs the assembly and GTPase activity of McrBC restriction complexes

Niu

Suzuki

Hosford

et al. 2020

Preprint

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13McrBC complexes are motor-driven nucleases functioning in bacterial self-defense by cleaving 14 foreign DNA. The GTP-specific AAA+ protein McrB powers translocation along DNA and its 15 hydrolysis activity is stimulated by its partner nuclease McrC. Here, we report cryo-EM 16 structures of Thermococcus gammatolerans McrB and McrBC, and E. coli McrBC. The McrB 17 hexamers, containing the necessary catalytic machinery for basal GTP hydrolysis, are 18 intrinsically asymmetric. This asymmetry directs McrC binding so that it engages a single active 19 site, where it then uses an arginine/lysine-mediated hydrogen-bonding network to reposition the 20 asparagine in the McrB signature motif for optimal catalytic function. While the two McrBC 21 complexes use different DNA-binding domains, these contribute to the same general GTP-22 recognition mechanism employed by all G proteins. Asymmetry also induces distinct inter-23 subunit interactions around the ring, suggesting a coordinated and directional GTP-hydrolysis 24 cycle. Our data provide novel insights into the conserved molecular mechanisms governing 25 McrB family AAA+ motors. upon research conducted at NE-CAT beamlines (24-ID-C and 24-ID-E) under the general user

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“…Numerous biochemical studies have demonstrated the role of an unstructured initiation or engagement region [17][18][19] , yet a substantial fraction of cellular proteasomal substrates appear to lack such flexible segments 20 , begging the question of how their degradation is initiated. While other unfoldases, like Cdc48/p97 may generate disordered regions 21,23,38 , it is also possible that for some proteins ubiquitin-mediated conformational changes are sufficient to expose the obligate unstructured segments. To test this hypothesis, we poly-ubiquitinated our panel of single-lysine barstar variants (Ubn-barstar) and assayed the proteasome's ability to recognize these substrates via its endogenous ubiquitin receptors and degrade them in an ATP-dependent manner (Fig.…”

Section: Substrate Ubiquitination Can Induce a Proteasome-engageable mentioning

confidence: 99%

Ubiquitination modulates a protein energy landscape site-specifically with consequences for proteasomal degradation

Carroll

Greene

Martin

et al. 2019

Preprint

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Cellular environments modulate protein energy landscapes to drive important biology, where small perturbations are consequential for biological signaling, allostery, and other vital processes. The energetic effects of ubiquitination are interesting due to its potential influence on degradation by the 26S proteasome, which requires intrinsically flexible or unstructured initiation regions that many known proteasome substrates lack. We generated proteins with natively attached, isopeptide-linked ubiquitin in structured domains to assess the energetic changes contributed by ubiquitin and how such changes manifest at the proteasome. Ubiquitination at sensitive sites destabilizes the native structure, and thereby increases the rate of degradation for substrates containing unstructured initiation regions. Importantly, this ubiquitination can even induce those requisite regions in well-folded proteins for proteasomal engagement. Our results indicate a biophysical role of site-specific ubiquitination as a potential regulatory mechanism for energy-dependent substrate degradation. Introduction:A protein's function and folding is defined by its energy landscape, a term which encompasses all the accessible conformations, their relative populations, and the rates of interconversion.This energy landscape is determined by a protein's amino-acid sequence and environment, and small changes in those parameters modulate this landscape. The phenotypic effects of these changes can range from undetectable to pathological 1-5 . Posttranslational modifications (PTMs) are one important environmental change that affect the energy landscape. Of the many PTMs that have been shown to affect protein structure and function 6 , the attachment of ubiquitin to lysine side chains is particularly interesting, as one of the most important roles for ubiquitination is to target proteins for degradation by the 26S proteasome.The ubiquitin-proteasome system (UPS) is responsible for the majority of protein turnover in eukaryotic cells. Ubiquitin is an 8.5 kDa protein appended to other proteins (substrates) through an isopeptide bond between its C-terminus and the amino group of lysines in the substrate.Ubiquitin itself contains seven lysines, such that additional ubiquitin molecules can be added to form chains of different lengths, linkage types, and topologies. The 26S proteasome is the executor of the UPS, using ubiquitin receptors to selectively bind poly-ubiquitinated substrates and degrade them in an ATP-dependent manner. The degradation activity resides in the proteasome's 20S core particle, whose proteolytic sites are sequestered inside a central cavity.Substrates are delivered to the 20S core particle largely through the 19S regulatory particle (RP), which caps one or both sides of the barrel-shaped 20S core. The RP recruits ubiquitinated substrates, mechanically unfolds them with its AAA+ (ATPase Associated with diverse cellular Activities) ATPase motor, and translocates the unstructured polypeptides into the core particle for cleavage into smal...

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Substrate processing by the Cdc48 ATPase complex is initiated by ubiquitin unfolding

Cited by 258 publications

References 64 publications

Seesaw Conformations of Npl4 in the Human p97 Complex and the Inhibitory Mechanism of a Disulfiram Derivative

Seesaw Conformations of Npl4 in the Human p97 Complex and the Inhibitory Mechanism of a Disulfiram Derivative

Structural asymmetry governs the assembly and GTPase activity of McrBC restriction complexes

Ubiquitination modulates a protein energy landscape site-specifically with consequences for proteasomal degradation

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