Structural Basis of Ubiquitin Recognition by the Deubiquitinating Protease USP2

Renatus, Martin; Parrado, S.G.; D’Arcy, Allan; Eidhoff, Ulf; Gerhartz, Bernd; Hassiepen, Ulrich; Pierrat, Benoı̂t; Riedl, Ralph; Vinzenz, Daniela; Worpenberg, Susanne; Kroemer, M.

doi:10.1016/j.str.2006.06.012

Cited by 189 publications

(250 citation statements)

References 39 publications

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“…In addition, it could allow binding of MdmX to USP2a in the correct orientation for deubiquitination or facilitate regulation of the catalytic activity of USP2a by MdmX. The crystal structure of the catalytic core of USP2 bound to ubiquitin has been determined (Renatus et al, 2006). The structure of USPs bound to ubiquitin has been likened to a hand.…”

Section: Discussionmentioning

confidence: 99%

“…The core of ubiquitin interacts with the fingers, palm and thumb of the USP2a hand, whereas the C-terminus of ubiquitin binds into a substrate cleft at the catalytic centre. Amino acids 403-503 do not include the active site Cys, QDE or Hisboxes but this region makes up four out of five of the 'fingers' which are involved in numerous contacts with the ubiquitin core (Renatus et al, 2006).…”

Section: Discussionmentioning

confidence: 99%

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MdmX is a substrate for the deubiquitinating enzyme USP2a

et al. 2009

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It has previously been shown that ubiquitin-specific protease 2a (USP2a) is a regulator of the Mdm2/p53 pathway. USP2a binds to Mdm2 and can deubiquitinate Mdm2 without reversing Mdm2-mediated p53 ubiquitination. Overexpression of USP2a causes accumulation of Mdm2 and promotes p53 degradation. We now show that MdmX is also a substrate for USP2a. MdmX associates with USP2a independently of Mdm2. Ectopic expression of wild-type USP2a but not a catalytic mutant prevents Mdm2-mediated degradation of MdmX. This correlates with the ability of wild-type USP2a to deubiquitinate MdmX. siRNA-mediated knockdown of USP2a in NTERA-2 testicular embryonal carcinoma cells and MCF7 breast cancer cells causes destabilization of MdmX and results in a decrease in MdmX protein levels, showing that endogenous USP2a participates in the regulation of MdmX stability. The therapeutic drug, cisplatin decreases MdmX protein expression. USP2a mRNA and protein levels were also reduced after cisplatin exposure. The magnitude and time course of USP2a downregulation suggests that the reduction in USP2a levels could contribute to the decrease in MdmX expression following treatment with cisplatin. Knockdown of USP2a increases the sensitivity of NTERA-2 cells to cisplatin, raising the possibility that suppression of USP2a in combination with cisplatin may be an approach for cancer therapy.

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Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

MdmX is a substrate for the deubiquitinating enzyme USP2a

et al. 2009

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“…Protein sample preparation (32), R 1ρ (12,33,34) and Carr-Purcell-Meiboom-Gill (CPMG) (35-37) experiments, and USP2 inhibition assays (32) were adapted from the cited work. Further details and other data analysis procedures are provided in SI Methods.…”

Section: Methodsmentioning

confidence: 99%

Allosteric switch regulates protein–protein binding through collective motion

Smith

Ban

Pratihar

et al. 2016

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

105

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Many biological processes depend on allosteric communication between different parts of a protein, but the role of internal protein motion in propagating signals through the structure remains largely unknown. Through an experimental and computational analysis of the ground state dynamics in ubiquitin, we identify a collective global motion that is specifically linked to a conformational switch distant from the binding interface. This allosteric coupling is also present in crystal structures and is found to facilitate multispecificity, particularly binding to the ubiquitin-specific protease (USP) family of deubiquitinases. The collective motion that enables this allosteric communication does not affect binding through localized changes but, instead, depends on expansion and contraction of the entire protein domain. The characterization of these collective motions represents a promising avenue for finding and manipulating allosteric networks.allostery | protein dynamics | concerted motion | relaxation dispersion | nuclear magnetic resonance I ntermolecular interactions are one of the key mechanisms by which proteins mediate their biological functions. For many proteins, these interactions are enhanced or suppressed by allosteric networks that couple distant regions together (1). The mechanisms by which these networks function are just starting to be understood (2-4), and many of the important details have yet to be uncovered. In particular, the role of intrinsic protein motion and kinetics remains particularly poorly characterized. A number of structural ensembles representing ubiquitin motion have been recently proposed (5-9). Additionally, it has been suggested that through motion at the binding interface, its free state visits the same conformations found in complex with its many binding partners (5, 10). However, it remains an unanswered question if the dynamics that enable this multispecificity are only clustered around the canonical binding interface or whether this motion is allosterically coupled to the rest of the protein, especially given the presence of motion at distal sites (11). ResultsTo answer this question and to provide a detailed structural picture of the underlying mechanism, we applied recently developed high-power relaxation dispersion (RD) experiments (12, 13) to both the backbone amide proton ( 1 H N ) and nitrogen ( 15 N) nuclei of ubiquitin. This survey yielded a nearly twofold increase in the number of nuclei where RD had been previously observed (11)(12)(13)(14) (from 17 to 31; Fig. 1A and Fig. S1). When fit individually, the full set of backbone and side-chain nuclei shows a consistent time scale of motion [exchange lifetime (τ ex ) = 55 μs; Fig. 1B]. Furthermore, the nuclei showing exchange are spread throughout the structure (Fig. 1C). Put together, these data suggest that the motions are not independent but share a common molecular mechanism.To determine whether the RD data could be modeled using a single collective motion, we developed a computational method to take a set of molecu...

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“…The study of USP-type DUB mechanism and regulation is complicated by their relatively poor activity: the catalytic domain of USP-type DUBs typically has an enzyme efficiency of 10 3 -10 5 M −1 ·s −1 and high micromolar substrate affinities, necessitating the use of covalent suicide "warheads" in structural studies (9). By contrast, UCH-type DUBs are often highly active, with enzyme efficiencies of up to 10 8 M −1 ·s −1 and affinity for ubiquitin in the nanomolar range (10,11).…”

mentioning

confidence: 99%

Conformational dynamics control ubiquitin-deubiquitinase interactions and influence in vivo signaling

Phillips¹,

Zhang²,

Cunningham³

et al. 2013

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

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Ubiquitin is a highly conserved eukaryotic protein that interacts with a diverse set of partners to act as a cellular signaling hub. Ubiquitin's conformational flexibility has been postulated to underlie its multifaceted recognition. Here we use computational and library-based means to interrogate core mutations that modulate the conformational dynamics of human ubiquitin. These ubiquitin variants exhibit increased affinity for the USP14 deubiquitinase, with concomitantly reduced affinity for other deubiquitinases. Strikingly, the kinetics of conformational motion are dramatically slowed in these variants without a detectable change in either the ground state fold or excited state population. These variants can be ligated into substrate-linked chains in vitro and in vivo but cannot solely support growth in eukaryotic cells. Proteomic analyses reveal nearly identical interaction profiles between WT ubiquitin and the variants but identify a small subset of altered interactions. Taken together, these results show that conformational dynamics are critical for ubiquitin-deubiquitinase interactions and imply that the fine tuning of motion has played a key role in the evolution of ubiquitin as a signaling hub.computational design | phage display | protein dynamics | protein-protein interactions | ubiquitin signaling

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Structural Basis of Ubiquitin Recognition by the Deubiquitinating Protease USP2

Cited by 189 publications

References 39 publications

MdmX is a substrate for the deubiquitinating enzyme USP2a

MdmX is a substrate for the deubiquitinating enzyme USP2a

Allosteric switch regulates protein–protein binding through collective motion

Conformational dynamics control ubiquitin-deubiquitinase interactions and influence in vivo signaling

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