A Conserved Asparagine in a Ubiquitin Conjugating Enzyme Positions the Substrate for Nucleophilic Attack

Jones, Walker M.; Davis, Aaron G.; Wilson, R. Hunter; Elliott, Katherine L.; Sumner, Isaiah

doi:10.26434/chemrxiv.7019579

Cited by 3 publications

(8 citation statements)

References 37 publications

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“…Based on the alignment in Fig 2A, the putative catalytic cysteine of UBC2 is C85, equivalent to C87 in S. cerevisiae UBC13 and C87 in human UBE2N (35). UBC2 also contains an HPN motif 10 amino acids Nterminal of C85 that may be involved in catalysis (28)(29)(30). All of the proteins in the UEV1 alignment ( Fig 2B) lack catalytic cysteine residues and HPN motifs, consistent with UEV1 being part of the noncatalytic UEV family of E2s (27).…”

Section: Ubc2 and Uev1 Are Orthologues Of Human Ube2n And Ube2v1/ube2v2mentioning

confidence: 72%

“…Furthermore, the HPN (His-Pro-Asn) motif, which is conserved in human E2-conjugating enzymes and which contains an asparagine residue that can be important for catalysis (28)(29)(30), was found to be partially or completely missing in UBC3, UBC6, UBC11, UBC14 and UEV1. A HECT domain alignment for L. mexicana HECT E3 ligases and 4 human HECT E3 ligases showed that HECTs 1-12 contained the conserved catalytic cysteine residue.…”

Section: Ubiquitination Gene Families In L Mexicanamentioning

confidence: 99%

“…Despite this, previous characterisation of the T. brucei orthologues of UBA2 and UBC9 by in vitro SUMOylation assays and UBA3 and UBC12 by affinity purification of Nedd8 lend further weight to their proposed functions (21,23). Of the putative ubiquitin E2s identified, 5 were missing the conserved asparagine residue thought to be important for E2 catalysis (28)(29)(30). UEV1, which also lacks the catalytic cysteine residue, is part of the non-catalytic ubiquitin E2 variant protein family.…”

Section: High Functional Conservation Of Human and Leishmania Enzymesmentioning

confidence: 99%

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Leishmaniadifferentiation requires ubiquitin conjugation mediated by a UBC2-UEV1 E2 complex

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Damianou

Wilkinson

et al. 2020

Preprint

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AbstractPost-translational modifications such as ubiquitination are important for orchestrating the cellular transformations that occur as the Leishmania parasite differentiates between its main morphological forms, the promastigote and amastigote. 2 E1 ubiquitin-activating (E1), 13 E2 ubiquitin-conjugating (E2), 79 E3 ubiquitin ligase (E3) and 20 deubiquitinating cysteine peptidase (DUB) genes can be identified in the Leishmania mexicana genome but, currently, little is known about the role of E1, E2 and E3 enzymes in this parasite. Bar-seq analysis of 23 E1, E2 and E3 null mutants generated in promastigotes using CRISPR-Cas9 revealed numerous loss-of-fitness phenotypes in promastigote to amastigote differentiation and mammalian infection. The E2s UBC1/CDC34, UBC2 and UEV1 and the HECT E3 ligase HECT2 are required for the successful transformation from promastigote to amastigote and UBA1b, UBC9, UBC14, HECT7 and HECT11 are required for normal proliferation during mouse infection. Null mutants could not be generated for the E1 UBA1a or the E2s UBC3, UBC7, UBC12 and UBC13, suggesting these genes are essential in promastigotes. X-ray crystal structure analysis of UBC2 and UEV1, orthologues of human UBE2N and UBE2V1/UBE2V2 respectively, reveal a heterodimer with a highly conserved interface, highlighting the importance of stable UBC2-UEV1 interaction in the function of this complex across diverse eukaryotes. Furthermore, recombinant L. mexicana E1 UBA1a can load ubiquitin onto UBC2, allowing UBC2-UEV1 to form K63-linked di-ubiquitin chains in vitro. Notably, UBC2 can also cooperate in vitro with human E3s RNF8 and BIRC2 to form non-K63-linked polyubiquitin chains, showing that UBC2 can facilitate ubiquitination independent of UEV1, but association of UBC2 with UEV1 inhibits this ability. Our study demonstrates the dual essentiality of UBC2 and UEV1 in the differentiation and intracellular survival of L. mexicana and shows that the interaction between these two proteins is crucial for regulation of their ubiquitination activity and function.Author summaryThe post-translational modification of proteins is key for allowing Leishmania parasites to transition between the different life cycle stages that exist in its insect vector and mammalian host. In particular, components of the ubiquitin system are important for the transformation of Leishmania from its insect (promastigote) to mammalian (amastigote) stage and normal infection in mice. However, little is known about the role of the enzymes that generate ubiquitin modifications in Leishmania. Here we characterise 28 enzymes of the ubiquitination pathway and show that many are required for life cycle progression or mouse infection by this parasite. Two proteins, UBC2 and UEV1, were selected for further study based on their importance in the promastigote to amastigote transition. We demonstrate that UBC2 and UEV1 form a heterodimer capable of carrying out ubiquitination and that the structural basis for this activity is conserved between Leishmania, Saccharomyces cerevisiae and humans. We also show that the interaction of UBC2 with UEV1 alters the nature of the ubiquitination activity performed by UBC2. Overall, we demonstrate the important role that ubiquitination enzymes play in the life cycle and infection process of Leishmania and explore the biochemistry underlying UBC2 and UEV1 function.

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Section: Ubc2 and Uev1 Are Orthologues Of Human Ube2n And Ube2v1/ube2v2mentioning

confidence: 72%

Section: Ubiquitination Gene Families In L Mexicanamentioning

confidence: 99%

Section: High Functional Conservation Of Human and Leishmania Enzymesmentioning

confidence: 99%

See 1 more Smart Citation

Leishmaniadifferentiation requires ubiquitin conjugation mediated by a UBC2-UEV1 E2 complex

Burge

Damianou

Wilkinson

et al. 2020

Preprint

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show abstract

“…Based on the alignment in Fig 2A, the putative catalytic cysteine of UBC2 is C85, equivalent to C87 in S. cerevisiae UBC13 and C87 in human UBE2N [39]. UBC2 also contains an HPN motif 10 amino acids N-terminal of C85 that may be involved in catalysis [31][32][33]. All of the proteins in the UEV1 alignment ( Fig 2B) lack catalytic cysteine residues and HPN motifs, consistent with UEV1 being part of the noncatalytic UEV family of E2s [30].…”

Section: Plos Pathogensmentioning

confidence: 72%

“…Similarly, an alignment of L. mexicana and human E2-conjugating enzymes showed conservation of the conserved catalytic cysteine in all L. mexicana E2s except UEV1 (S1B Fig), suggesting the latter is a non-catalytic ubiquitin E2 variant [30]. Furthermore, the HPN (His-Pro-Asn) motif, which is conserved in human E2-conjugating enzymes and which contains an asparagine residue that can be important for catalysis [31][32][33], was found to be partially or completely missing in UBC3, UBC6, UBC11, UBC14 and UEV1. A HECT domain alignment for L. mexicana HECT E3 ligases and 4 human HECT E3 ligases showed that HECTs 1-12 contained the conserved catalytic cysteine residue.…”

Section: Plos Pathogensmentioning

confidence: 89%

Leishmania differentiation requires ubiquitin conjugation mediated by a UBC2-UEV1 E2 complex

et al. 2020

View full text Add to dashboard Cite

Post-translational modifications such as ubiquitination are important for orchestrating the cellular transformations that occur as the Leishmania parasite differentiates between its main morphological forms, the promastigote and amastigote. 2 E1 ubiquitin-activating (E1), 13 E2 ubiquitin-conjugating (E2), 79 E3 ubiquitin ligase (E3) and 20 deubiquitinating cysteine peptidase (DUB) genes can be identified in the Leishmania mexicana genome but, currently, little is known about the role of E1, E2 and E3 enzymes in this parasite. Bar-seq analysis of 23 E1, E2 and HECT/RBR E3 null mutants generated in promastigotes using CRISPR-Cas9 revealed numerous loss-of-fitness phenotypes in promastigote to amastigote differentiation and mammalian infection. The E2s UBC1/CDC34, UBC2 and UEV1 and the HECT E3 ligase HECT2 are required for the successful transformation from promastigote to amastigote and UBA1b, UBC9, UBC14, HECT7 and HECT11 are required for normal proliferation during mouse infection. Of all ubiquitination enzyme null mutants examined in the screen, Δubc2 and Δuev1 exhibited the most extreme loss-of-fitness during differentiation. Null mutants could not be generated for the E1 UBA1a or the E2s UBC3, UBC7, UBC12 and UBC13, suggesting these genes are essential in promastigotes. X-ray crystal structure analysis of UBC2 and UEV1, orthologues of human UBE2N and UBE2V1/UBE2V2 respectively, reveal a heterodimer with a highly conserved structure and interface. Furthermore, recombinant L. mexicana UBA1a can load ubiquitin onto UBC2, allowing UBC2-UEV1 to form K63-linked di-ubiquitin chains in vitro. Notably, UBC2 can cooperate in vitro with human E3s RNF8 and BIRC2 to form non-K63-linked polyubiquitin chains, showing that UBC2 can facilitate ubiquitination independent of UEV1, but association of UBC2 with UEV1 inhibits this ability. Our study demonstrates the dual essentiality of UBC2 and UEV1 in the differentiation and intracellular survival of L. mexicana and shows that the interaction between these two proteins is crucial for regulation of their ubiquitination activity and function.

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Functional conservation and divergence of the helix‐turn‐helix motif of E2 ubiquitin‐conjugating enzymes

et al. 2021

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Polyubiquitination by E2 and E3 enzymes is crucial to cell cycle control, epigenetic regulation, and development. The hallmark of the E2 family is the ubiquitin (Ub)-conjugating (UBC) domain that forms a dynamic thioester conjugate with ubiquitin (E2~Ub). Numerous studies have focused on E2 surfaces, such as the Nterminal and crossover helices, that directly interact with an E3 or the conjugated ubiquitin to stabilize the active, "closed" state of the E2~Ub. However, it remains unclear how other E2 surfaces regulate ubiquitin transfer. Here, we demonstrate the helix-turnhelix (HTH) motif of the UBC tunes the intrinsic polyubiquitination activity through distinct functions in different E2s. Interestingly, the E2 HTH motif is repurposed in UBE2S and UBE2R2 to interact with the conjugated or acceptor ubiquitin, respectively, modulating ubiquitin transfer. Furthermore, we propose that Anaphase-Promoting Complex/Cyclosome binding to the UBE2S HTH reduces the conformational space of the flexible E2~Ub, demonstrating an atypical E3-dependent activation mechanism. Altogether, we postulate the E2 HTH motif evolved to provide new functionalities that can be harnessed by E3s and permits additional regulation to facilitate specific E2-E3-mediated polyubiquitination.

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A Conserved Asparagine in a Ubiquitin Conjugating Enzyme Positions the Substrate for Nucleophilic Attack

Cited by 3 publications

References 37 publications

Leishmaniadifferentiation requires ubiquitin conjugation mediated by a UBC2-UEV1 E2 complex

Leishmaniadifferentiation requires ubiquitin conjugation mediated by a UBC2-UEV1 E2 complex

Leishmania differentiation requires ubiquitin conjugation mediated by a UBC2-UEV1 E2 complex

Functional conservation and divergence of the helix‐turn‐helix motif of E2 ubiquitin‐conjugating enzymes

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