Crystal Structure of UBA2ufd-Ubc9: Insights into E1-E2 Interactions in Sumo Pathways

Wang, Jing; Taherbhoy, Asad M.; Hunt, Harold W.; Seyedin, Steven N.; Miller, David W.; Miller, Darcie J.; Huang, Danny T.; Schulman, Brenda A.

doi:10.1371/journal.pone.0015805

Cited by 42 publications

(52 citation statements)

References 57 publications

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“…For this analysis, we expressed and purified the C-terminal ubiquitin-fold domains of human and P. falciparum Uba2 (Uba2 ufd ), which are predicted to mediate interactions with Ubc9 (16,19). Consistent with this prediction, we observed endothermic binding between human Ubc9 and human Uba2 , and ⌬S ϭ 36 Ϯ 0.9 cal mol Ϫ1 K Ϫ1 (mean Ϯ S.D.…”

Section: Resultssupporting

confidence: 60%

“…5, b-d). Based on crystallographic analysis of S. cerevisiae E1 and E2 interactions (19), the ␣1 helix and the ␤1-␤2 loop are predicted to form the E1 interaction interface. Of particular interest, several residues within this region of PfUbc9, including Lys-5 and Lys-6, Ala-13 and Glu-14, and Pro-29 and Lys-34 have diverged from residues in HsUbc9 (Fig.…”

Section: Resultsmentioning

confidence: 99%

“…The solved structure of a complex between S. cerevisiae Uba2 ufd (ScUba2 ufd ) and ScUbc9 was used to guide protein alignment and build homology models (19). Results from this analysis indicated that the E1 and E2 enzymes have co-evolved to maintain surface residues compatible for interaction.…”

Section: Resultsmentioning

confidence: 99%

“…1b). Detailed biochemical and structural studies of the E1 and E2 enzymes from human and yeast have been reported, and are available for guiding comparative studies (15)(16)(17)(18)(19).…”

mentioning

confidence: 99%

See 3 more Smart Citations

Identification of Biochemically Distinct Properties of the Small Ubiquitin-related Modifier (SUMO) Conjugation Pathway in Plasmodium falciparum

Reiter

Mukhopadhyay

Zhang

et al. 2013

Journal of Biological Chemistry

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Background:The sumoylation pathway is conserved in Plasmodium falciparum. Results: The small ubiquitin-related modifier (SUMO) E1 and E2 enzymes are not functionally interchangable between humans and the malaria parasite, P. falciparum. Conclusion: P. falciparum E1 and E2 interactions have significantly diverged from humans. Significance: Divergent E1 and E2 interaction could be exploited for the design of parasite specific inhibitors.

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Section: Resultssupporting

confidence: 60%

Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

“…1b). Detailed biochemical and structural studies of the E1 and E2 enzymes from human and yeast have been reported, and are available for guiding comparative studies (15)(16)(17)(18)(19).…”

mentioning

confidence: 99%

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Identification of Biochemically Distinct Properties of the Small Ubiquitin-related Modifier (SUMO) Conjugation Pathway in Plasmodium falciparum

Reiter

Mukhopadhyay

Zhang

et al. 2013

Journal of Biological Chemistry

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“…In similarity to E2 enzymes in the ubiquitin proteasome pathway, crayfish UBC9 contains a core ubiquitin-conjugating catalytic domain (UBCc) with a conserved Cys93 (33). However, SUMOylation solely utilizes the E2 UBC9, whereas the ubiquitination pathway uses diverse E2s.…”

Section: Discussionmentioning

confidence: 99%

SUMO-Conjugating Enzyme E2 UBC9 Mediates Viral Immediate-Early Protein SUMOylation in Crayfish To Facilitate Reproduction of White Spot Syndrome Virus

Chen

Wang

Zhao

et al. 2013

J Virol

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Successful viruses have evolved superior strategies to escape host defenses or exploit host biological pathways. Most of the viral immediate-early (ie) genes are essential for viral infection and depend solely on host proteins; however, the molecular mechanisms are poorly understood. In this study, we focused on the modification of viral IE proteins by the crayfish small ubiquitinrelated modifier (SUMO) and investigated the role of SUMOylation during the viral life cycle. SUMO and SUMO ubiquitin-conjugating enzyme 9 (UBC9) involved in SUMOylation were identified in red swamp crayfish (Procambarus clarkii). Both SUMO and UBC9 were upregulated in crayfish challenged with white spot syndrome virus (WSSV). Replication of WSSV genes increased in crayfish injected with recombinant SUMO or UBC9, but injection of mutant SUMO or UBC9 protein had no effect. Subsequently, we analyzed the mechanism by which crayfish SUMOylation facilitates WSSV replication. Crayfish UBC9 bound to all three WSSV IE proteins tested, and one of these IE proteins (WSV051) was covalently modified by SUMO in vitro. The expression of viral ie genes was affected and that of late genes was significantly inhibited in UBC9-silenced or SUMO-silenced crayfish, and the inhibition effect was rescued by injection of recombinant SUMO or UBC9. The results of this study demonstrate that viral IE proteins can be modified by crayfish SUMOylation, prompt the expression of viral genes, and ultimately benefit WSSV replication. Understanding of the mechanisms by which viruses exploit host components will greatly improve our knowledge of the virus-host "arms race" and contribute to the development of novel methods against virulent viruses.

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Insights Into the Allosteric Inhibition of the SUMO E2 Enzyme Ubc9

et al. 2016

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Conjugation of the small ubiquitin-like modifier (SUMO) to protein substrates is an important disease-associated posttranslational modification, although few inhibitors of this process are known. Herein, we report the discovery of an allosteric small-molecule binding site on Ubc9, the sole SUMO E2 enzyme.A nX -rayc rystallographic screen was used to identify two distinct small-molecule fragments that bind to Ubc9 at as ite distal to its catalytic cysteine.T hese fragments and related compounds inhibit SUMO conjugation in biochemical assays with potencies of 1.9-5.8 mm.Mechanistic and biophysical analyses,c oupled with molecular dynamics simulations,p oint toward ligand-induced rigidification of Ubc9 as amechanism of inhibition.The posttranslational modification of protein substrates with as mall ubiquitin-like modifier (SUMO) tag occurs through at ightly regulated E1/E2/E3 enzymatic cascade and modulates ab road variety of cellular functions.[1] Ubc9 is the only E2 enzyme involved in the conjugation of all three SUMO isoforms to many diverse substrates within the proteome.As acentral enzyme in the SUMOylation cycle,Ubc9 is required for normal embryonic development, and it is dysregulated in av ariety of disease states such as cancer [2] and ischemia.[3]Ubc9 has been suggested as ap otential anticancer target for small-molecule inhibitors,n otably for MYC-driven [4] and RAS/Raf-driven [5] cancers,a sw ell as multiple myeloma.[6]Despite its relevance to disease,i th as been challenging to identify small molecules that modulate the function of Ubc9.In related ubiquitin-like signaling pathways,inhibitors of E1 [7] and E3 [8] enzymes are now clinically used as anticancer chemotherapeutics.Bycontrast, very few reversible inhibitors of any of the approximately 40 known ubiquitin and ubiquitin-like E2 conjugating enzymes have been discovered, [9] only one of which has been characterized in complex with the protein.[10] Thus,n ew experimentally validated chemical inhibitors of Ubc9 would provide substantial insight into targeting this important enzyme,a nd potentially E2 enzymes in general.One powerful approach to identify ligands for challenging protein targets is fragment-based inhibitor discovery.[11] In general, fragment-based approaches leverage sensitive techniques such as NMR, thermal shift, surface plasmon resonance (SPR), or X-ray crystallography to identify lowmolecular-weight ligands that bind weakly but specifically to target proteins.[12] Weak leads identified through fragmentbased approaches can provide excellent starting points for the development of highly active inhibitors,e ven in cases where the targets are considered to be challenging,such as proteinprotein interactions or so-called "undruggable" targets. [13] As part of al arger program aimed toward identifying chemical inhibitors of Ubc9, we elected to pursue an X-ray crystallographic fragment screening strategy (see the Supporting Information).[14] Ubc9 crystals diffracting to aresolution of 1.12 ( PDB ID:5 F6E) were soaked with as eries ...

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Crystal Structure of UBA2ufd-Ubc9: Insights into E1-E2 Interactions in Sumo Pathways

Cited by 42 publications

References 57 publications

Identification of Biochemically Distinct Properties of the Small Ubiquitin-related Modifier (SUMO) Conjugation Pathway in Plasmodium falciparum

Identification of Biochemically Distinct Properties of the Small Ubiquitin-related Modifier (SUMO) Conjugation Pathway in Plasmodium falciparum

SUMO-Conjugating Enzyme E2 UBC9 Mediates Viral Immediate-Early Protein SUMOylation in Crayfish To Facilitate Reproduction of White Spot Syndrome Virus

Insights Into the Allosteric Inhibition of the SUMO E2 Enzyme Ubc9

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