Backbone and ILV side-chain NMR resonance assignments of the catalytic domain of human deubiquitinating enzyme USP7

Valles, Gabrielle J.; Pozhidaeva, Alexandra; Korzhnev, Dmitry M.

doi:10.1007/s12104-022-10079-2

Cited by 4 publications

(3 citation statements)

References 38 publications

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“…Previously reported NMR resonance assignments of the backbone and ILV methyl groups were used [35] (BMRB ID: 51188). All Carr-Purcell-Meiboom-Gill relaxation dispersion (CPMG RD) NMR experiments were performed on an 800 MHz ( 1 H) Agilent VNMRS spectrometer equipped with a room-temperature HCN Z-gradient probe at 30°C.…”

Section: Methodsmentioning

confidence: 99%

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Activation Dynamics of Ubiquitin Specific Protease 7

Valles

Jaiswal

Korzhnev

et al. 2023

Preprint

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Ubiquitin-specific protease 7 (USP7) is a deubiquitinating enzyme responsible for the regulation of key human oncoproteins and tumor suppressors including Mdm2 and p53, respectively. Unlike other members of the USP family of proteases, the isolated catalytic domain of USP7 adopts an enzymatically inactive conformation that has been well characterized using X-ray crystallography. The catalytic domain also samples an active conformation, which has only been captured upon USP7 substrate-binding. Here, we utilized CPMG NMR relaxation dispersion studies to observe the dynamic motions of USP7 in solution. Our results reveal that the catalytic domain of USP7 exchanges between two distinct conformations, the inactive conformation populated at 95% and the active conformation at 5%. The largest structural changes are localized within functionally important regions of the enzyme including the active site, the ubiquitin-binding fingers, and the allosteric helix of the enzyme, suggesting that USP7 can adopt its active conformation in the absence of a substrate. Furthermore, we show that the allosteric L299A activating mutation disturbs this equilibrium, slows down the exchange, and increases the residence time of USP7 in its active conformation, thus, explaining the elevated activity of the mutant. Overall, this work shows that the isolated USP7 catalytic domain pre-samples its “invisible” active conformation in solution, which may contribute to its activation mechanism.

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Section: Methodsmentioning

confidence: 99%

“…[37] and analyzed in Sparky [38], as implemented in the NMRbox platform [39], using previously reported the backbone and ILV side-chain resonance assignments [35] for the WT. The C223A and L299A USP-CD resonances were assigned based on their similarity to the WT where possible.…”

Section: Methodsmentioning

confidence: 99%

Activation Dynamics of Ubiquitin Specific Protease 7

Valles

Jaiswal

Korzhnev

et al. 2023

Preprint

Self Cite

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“…In-depth experimental analysis of smaller proteins has shown the pronounced role of dynamics in allosteric networks [26,[28][29][30][31][32]. However, due to the somewhat challenging size of the USP domain (>40 kDa), NMR studies have, to date, only been pursued for USP7, focusing on ligand and inhibitor binding [33][34][35][36][37], leaving the loop dynamics understudied.…”

Section: Introductionmentioning

confidence: 99%

Dynamic regions allosterically connect the USP14 active site with the proteasome interaction surface

Salomonsson,

Sjöstrand,

Eskilson

et al. 2024

Preprint

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Ubiquitin-specificprotease 14 (USP14), is a member of the USP family responsible for the catalytic removal of ubiquitin (Ub) from proteins directed to the proteasome, implicated in the pathogenesis of neurodegeneration and cancer. Crystallography and cryo-EM analysis have identified loop regions crucial for the deubiquitinase activity of USP14, specifically those involved in Ub and proteasome binding. However, the structural changes in USP14 upon ligand binding to these regions are minimal, indicating significant yet uncharacterized dynamic contributions to its function. In this study, through structural and dynamical NMR experiments and functional evaluation, we demonstrate that small mutations designed to impact Ub binding and catalytic activity without disturbing the USP structure display both local and long-range effects. The affected residues connect the active site and the Ub binding region with the proteasome interaction surface through a network of loops, which show varied dynamics on the ps-ms time scale. Collectively, our findings experimentally reveal different aspects of dynamic connections within USP14, suggesting the presence of allosteric networks that link enzyme activity with regulatory function. The novel concept that USP14 allosteric networks are pre-existing, coupled, and activated by regulatory interactions with the USP fold, could be crucial to future targeted drug design.

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