The Intrinsic Affinity between E2 and the Cys Domain of E1 in Ubiquitin-like Modifications

Wang, Jiang-Hai; Hu, Weidong; Cai, Shuqun; Lee, Brian; Song, Jing; Chen, Yuan

doi:10.1016/j.molcel.2007.05.023

Cited by 59 publications

(74 citation statements)

References 29 publications

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“…This surface is distinct from that required for the interaction of UBC9 with the Sae2 E1-activating enzyme and the catalytic site (Wang et al, 2007), or interaction with the CKxE/D motif in a target substrate (Lin et al, 2002). Thus, E1A should not affect the ability of UBC9 to accept activated SUMO from Sae2 or recognize and modify a target substrate, which agrees with our observation that E1A does not affect in vitro monoSUMOylation (Supplementary Figure S3).…”

Section: Discussionsupporting

confidence: 88%

Identification of a molecular recognition feature in the E1A oncoprotein that binds the SUMO conjugase UBC9 and likely interferes with polySUMOylation

et al. 2010

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Hub proteins have central roles in regulating cellular processes. By targeting a single cellular hub, a viral oncogene may gain control over an entire module in the cellular interaction network that is potentially comprised of hundreds of proteins. The adenovirus E1A oncoprotein is a viral hub that interacts with many cellular hub proteins by short linear motifs/molecular recognition features (MoRFs). These interactions transform the architecture of the cellular protein interaction network and virtually reprogram the cell. To identify additional MoRFs within E1A, we screened portions of E1A for their ability to activate yeast pseudohyphal growth or differentiation. This identified a novel functional region within E1A conserved region 2 comprised of the sequence EVIDLT. This MoRF is necessary and sufficient to bind the N-terminal region of the SUMO conjugase UBC9, which also interacts with SUMO noncovalently and is involved in polySUMOylation. Our results suggest that E1A interferes with polySUMOylation, but not with monoSUMOylation. These data provide the first insight into the consequences of the interaction of E1A with UBC9, which was initially described in 1996. We further demonstrate that polySUMOylation regulates pseudohyphal growth and promyelocytic leukemia body reorganization by E1A. In conclusion, the interaction of the E1A oncogene with UBC9 mimics the normal binding between SUMO and UBC9 and represents a novel mechanism to modulate polySUMOylation.

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Section: Discussionsupporting

confidence: 88%

Identification of a molecular recognition feature in the E1A oncoprotein that binds the SUMO conjugase UBC9 and likely interferes with polySUMOylation

et al. 2010

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“…This fragment was cloned into the NdeI-XhoI sites of vector pET28a to express the E1 Ubl domain protein with a C-terminal His 6 tag. Site-directed mutagenesis, protein purification, and preparation of stable isotope-labeled protein samples for NMR experiments were carried out as described previously (11).…”

Section: Methodsmentioning

confidence: 99%

“…The steady-state kinetic assay for RanGAP1⅐SUMO complex formation under E1-limiting conditions was described previously (11,19), except that E1 concentration was 1 M for the GGG mutant and 400 nM for the 484⌬ mutant, respectively. The percentages of E1 enzymes that are active are 30 and 37% for GGG and 484D, respectively, judged by the percentage of E1 that can form thioester conjugates with SUMO.…”

Section: C]glucose and [mentioning

confidence: 99%

“…During the transfer of Ublp from E1 to E2, the E2 enzyme is recruited to the E1-SUMO thioester conjugate by binding to multiple sites on E1, including the Cys domain (the domain containing the active Cys residue) and the ubiquitin-like (Ubl) domain (10,11). Among these multiple binding sites, the Ubl domain has the highest binding affinity, and thus it is the key E2-binding site.…”

mentioning

confidence: 99%

“…First, the E2-binding surface on the Cys domain of E1 contains an extended and flexible loop (11). Second, the E2-binding surface on the Ubl domains of SUMO and NEDD8 E1s either contains missing segments or has segments with unusually large B-factors in x-ray crystal structures (12,13), suggesting that conformational flexibility also exists at the E2-binding surface of the Ubl domain.…”

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confidence: 99%

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Conformational Transition Associated with E1-E2 Interaction in Small Ubiquitin-like Modifications

Wang¹,

Lee²,

Cai³

et al. 2009

Journal of Biological Chemistry

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Ubiquitin-like modifications regulate nearly every aspect of cellular functions. A key step in these modifications is the recognition of the carrier enzyme (E2) by the activating enzyme (E1). In this study, we have found that a critical E2-binding surface on the E1 of the small ubiquitin-like modifier has unusually high populations in both ordered and disordered states. Upon binding the E2, the disordered state is converted to the ordered state, which resembles the structure of the bound conformation, providing a mechanism to resolve the "Levinthal Paradox" search problem in a folding-upon-binding process. The significance of the folding-unfolding equilibrium is shown by the loss of functions of the mutations that shift the equilibrium to the folded state. This study highlights the importance of conformational flexibility in the molecular recognition event.The conjugation of ubiquitin or ubiquitin-like proteins to other cellular proteins is one of the most important mechanisms in regulating cellular functions in eukaryotic systems and is broadly involved in controlling the life spans, trafficking, and functions of a wide range of proteins (1-4). Among the ubiquitin-like modifiers, the small ubiquitin-like modifier (SUMO) 5 is the most studied, and its modification regulates many essential functions, such as gene transcription, hormone response, DNA repair, and nuclear import (5-7). Multiple enzymes are required for post-translational modifications by ubiquitin or ubiquitin-like proteins (1,8). A ubiquitin-like protein (Ublp) is first activated by E1. The E1 required for SUMO modification is a tight heterodimer of two proteins, known as SUMO activation enzymes 1 and 2 (SAE1 and SAE2), which are homologous to the N-terminal and C-terminal portions of the ubiquitin E1, respectively (9). E1 catalyzes the adenylation of the C-terminal carboxyl group of the Ublp, and it then forms a thioester bond between the -SH group of the active site Cys residue and the C-terminal carboxyl group of the Ublp. The Ublp is then transferred to E2 (known as Ubc9 in the SUMO pathway), forming a thioester bond with the -SH group of the active site Cys residue. In the final step, the Ublp is attached to target proteins by forming an isopeptide bond between its C-terminal carboxyl group and the ⑀-amino group of a Lys residue on the target protein. This step generally requires another enzyme, isopeptide ligase.During the transfer of Ublp from E1 to E2, the E2 enzyme is recruited to the E1-SUMO thioester conjugate by binding to multiple sites on E1, including the Cys domain (the domain containing the active Cys residue) and the ubiquitin-like (Ubl) domain (10, 11). Among these multiple binding sites, the Ubl domain has the highest binding affinity, and thus it is the key E2-binding site. The multivalent interaction produces high affinity binding between E1 and E2, which accounts for the efficiency of E1 at low concentrations. At the same time, the low to medium affinity of each of the individual interactions allows fast turnover of the enzym...

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Biochemical Analysis of Protein SUMOylation

Alontaga

Bobkova

Chen

2012

CP Molecular Biology

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SUMOylation, the covalent attachment of Small Ubiquitin-like MOdifier (SUMO) polypeptides to other proteins, is among the most important post-translational modifications that regulate the functional properties of a large number of proteins. SUMOylation is broadly involved in cellular processes such as gene transcription, hormone response, signal transduction, DNA repair and nuclear transport. SUMO modification has also been implicated in the pathogenesis of human diseases, such as cancer, neurodegenerative disorders and viral infection. Attachment of a SUMO protein to another protein is carried out in multiple steps catalyzed by three enzymes. This unit describes and discusses the in vitro biochemical methods used for investigating each step of the SUMOylation process. In addition, a high throughput screening protocol is included for the identification of inhibitors of SUMOylation.

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The Intrinsic Affinity between E2 and the Cys Domain of E1 in Ubiquitin-like Modifications

Cited by 59 publications

References 29 publications

Identification of a molecular recognition feature in the E1A oncoprotein that binds the SUMO conjugase UBC9 and likely interferes with polySUMOylation

Identification of a molecular recognition feature in the E1A oncoprotein that binds the SUMO conjugase UBC9 and likely interferes with polySUMOylation

Conformational Transition Associated with E1-E2 Interaction in Small Ubiquitin-like Modifications

Biochemical Analysis of Protein SUMOylation

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