Insights into Ubiquitin Product Release in Hydrolysis Catalyzed by the Bacterial Deubiquitinase SdeA

Sheedlo, M.J.; Kenny, Sebastian; Podkorytov, Ivan S.; Brown, Kwame; Ma, Jia; Iyer, Shalini; Hewitt, Chad S.; Arbough, Trent; Mikhailovskii, Oleg; Flaherty, Daniel P.; Wilson, Mark A.; Skrynnikov, Nikolai R.; Das, Chittaranjan

doi:10.1021/acs.biochem.0c00760

Cited by 5 publications

(5 citation statements)

References 56 publications

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“…While the previous structures of Ub bound to SdeA DUB (Sheedlo et al, 2015(Sheedlo et al, , 2021 had many similarities to the disulfide-linked structure obtained in this study, this new structure shows a different arrangement of the catalytic cysteine and some nearby residues in the DUB active site (Fig. 2d and Supplementary Figs.…”

Section: Discussionsupporting

confidence: 61%

“…Two previously crystallized complexes of bacterial DUBs from pathogenic organisms were subjected to crystallization as their Ub G76C complex, allowing an assessment of the disulfide strategy. While the SdeA DUB, an effector protein from the arsenal of Legionella pneumophila (Sheedlo et al, 2015(Sheedlo et al, , 2021, reacted with the Ub cysteine in the expected manner showing Ub binding at the S1 site, the effector DUB from Orientia tsutsugamushi (OtDUB; Berk et al, 2020) preferentially reacted with a noncatalytic cysteine.…”

Section: Introductionmentioning

confidence: 99%

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Cocrystallization of ubiquitin–deubiquitinase complexes through disulfide linkage

Negron Teron,

Das

2023

Acta Cryst Sect D Struct Biol

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Structural characterization of the recognition of ubiquitin (Ub) by deubiquitinases (DUBs) has largely relied on covalent complexation of the DUB through its catalytic cysteine with a Ub C-terminal electrophile. The Ub electrophiles are accessed through intein chemistry in conjunction with chemical synthesis. Here, it was asked whether DUB–Ub covalent complexes could instead be accessed by simpler disulfide chemistry using a Ub cysteine mutant in which the last glycine has been replaced with a cysteine. The Ub cysteine mutant displayed a wide variability in disulfide formation across a panel of eukaryotic and prokaryotic DUBs, with some showing no detectable reaction while others robustly produced a disulfide complex. Using this approach, two disulfide-linked ubiquitin-bound complexes were crystallized, one involving the Legionella pneumophila effector SdeA DUB and the other involving the Orientia effector OtDUB. These DUBs had previously been crystallized in Ub-bound forms using the C-terminal electrophile strategy and noncovalent complexation, respectively. While the disulfide-linked SdeA DUB–Ub complex crystallized as expected, in the OtDUB complex the disulfide bond to the Ub mutant involved a cysteine that differed from the catalytic cysteine. Disulfide formation with the SdeA DUB catalytic cysteine was accompanied by local distortion of the helix carrying the active-site cysteine, whereas OtDUB reacted with the Ub mutant using a surface-exposed cysteine.

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Section: Discussionsupporting

confidence: 61%

Section: Introductionmentioning

confidence: 99%

Cocrystallization of ubiquitin–deubiquitinase complexes through disulfide linkage

Negron Teron,

Das

2023

Acta Cryst Sect D Struct Biol

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“…3 ). These two amino acids are part of the L RG G binding motif, a cleavage site recognized by deubiquitinating enzymes (DUBs) [ 26 – 28 ]. DUBs are responsible for both removing polyubiquitin chains from substrate proteins and generating free ubiquitin monomers, playing a crucial role in the regulation of the ubiquitin–proteasome system.…”

Section: Resultsmentioning

confidence: 99%

The unusual gene architecture of polyubiquitin is created by dual-specific splice sites

Duan,

Mooney,

Buerer

et al. 2024

Genome Biol

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Background The removal of introns occurs through the splicing of a 5′ splice site (5′ss) with a 3′ splice site (3′ss). These two elements are recognized by distinct components of the spliceosome. However, introns in higher eukaryotes contain many matches to the 5′ and 3′ splice-site motifs that are presumed not to be used. Results Here, we find that many of these sites can be used. We also find occurrences of the AGGT motif that can function as either a 5′ss or a 3′ss—previously referred to as dual-specific splice sites (DSSs)—within introns. Analysis of the Sequence Read Archive reveals a 3.1-fold enrichment of DSSs relative to expectation, implying synergy between the ability to function as a 5′ss and 3′ss. Despite this suggested mechanistic advantage, DSSs are 2.7- and 4.7-fold underrepresented in annotated 5′ and 3′ splice sites. A curious exception is the polyubiquitin gene UBC, which contains a tandem array of DSSs that precisely delimit the boundary of each ubiquitin monomer. The resulting isoforms splice stochastically to include a variable number of ubiquitin monomers. We found no evidence of tissue-specific or feedback regulation but note the 8.4-fold enrichment of DSS-spliced introns in tandem repeat genes suggests a driving role in the evolution of genes like UBC. Conclusions We find an excess of unannotated splice sites and the utilization of DSSs in tandem repeats supports the role of splicing in gene evolution. These findings enhance our understanding of the diverse and complex nature of the splicing process.

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“…Mono-ubiquitin variants were purified from a pRSET-A vector as previously described [ 20 , 31 ] and summarized below. The untagged WT-Ub pRSET-A vector construct was obtained from Dr. Das.…”

Section: Methodsmentioning

confidence: 99%

Rational Development and Characterization of a Ubiquitin Variant with Selectivity for Ubiquitin C-Terminal Hydrolase L3

2022

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There is currently a lack of reliable methods and strategies to probe the deubiquitinating enzyme UCHL3. Current small molecules reported for this purpose display reduced potency and selectivity in cellular assays. To bridge this gap and provide an alternative approach to probe UCHL3, our group has carried out the rational design of ubiquitin-variant activity-based probes with selectivity for UCHL3 over the closely related UCHL1 and other DUBs. The approach successfully produced a triple-mutant ubiquitin variant activity-based probe, UbVQ40V/T66K/V70F-PRG, that was ultimately 20,000-fold more selective for UCHL3 over UCHL1 when assessed by rate of inactivation assays. This same variant was shown to selectively form covalent adducts with UCHL3 in MDA-MB-231 breast cancer cells and no reactivity toward other DUBs expressed. Overall, this study demonstrates the feasibility of the approach and also provides insight into how this approach may be applied to other DUB targets.

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Insights into Ubiquitin Product Release in Hydrolysis Catalyzed by the Bacterial Deubiquitinase SdeA

Cited by 5 publications

References 56 publications

Cocrystallization of ubiquitin–deubiquitinase complexes through disulfide linkage

Cocrystallization of ubiquitin–deubiquitinase complexes through disulfide linkage

The unusual gene architecture of polyubiquitin is created by dual-specific splice sites

Rational Development and Characterization of a Ubiquitin Variant with Selectivity for Ubiquitin C-Terminal Hydrolase L3

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