Browse
Submit Data
Databases
API
Help

Dataset Information

23 Views

0 Connections

0 Citations

0 Reanalyses

0 Downloads

Omics score: 0

Conformational transition associated with E1-E2 interaction in small ubiquitin-like modifications.

ABSTRACT: 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.

SUBMITTER: Wang J

PROVIDER: S-EPMC2740459 | biostudies-literature | 2009 Jul

REPOSITORIES: biostudies-literature

ACCESS DATA

Json Xml

Publications

Conformational transition associated with E1-E2 interaction in small ubiquitin-like modifications.

Wang Jianghai J Lee Brian B Cai Sheng S Fukui Lisa L Hu Weidong W Chen Yuan Y

The Journal of biological chemistry 20090514 30

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 conform ...[more]

PMID: 19443651

Similar Datasets

Structure of a ubiquitin E1-E2 complex: insights to E1-E2 thioester transfer.

Project description:Ubiquitin (Ub) conjugation is initiated by an E1 enzyme that catalyzes carboxy-terminal Ub adenylation, thioester bond formation to a catalytic cysteine in the E1 Cys domain, and thioester transfer to a catalytic cysteine in E2 conjugating enzymes. How the E1 and E2 active sites come together during thioester transfer and how Ub E1 interacts with diverse Ub E2s remains unclear. Here we present a crystal structure of a Ub E1-E2(Ubc4)/Ub/ATP·Mg complex that was stabilized by induction of a disulfide bond between the E1 and E2 active sites. The structure reveals combinatorial recognition of the E2 by the E1 ubiquitin-fold domain (UFD) and Cys domain and mutational analysis, coupled with thioester transfer assays with E1, Ubc4, and other Ub E2s, show that both interfaces are important for thioester transfer. Comparison to a Ub E1/Ub/ATP·Mg structure reveals conformational changes in the E1 that bring the E1 and E2 active sites together.

| S-EPMC3625138 | biostudies-literature

The intrinsic affinity between E2 and the Cys domain of E1 in ubiquitin-like modifications.

Project description:Ubiquitin-like modifications, which are carried out by similar biochemical mechanisms, regulate nearly every aspect of cellular function. Despite the recent advancements in characterizing their enzymology, our knowledge about the dynamic processes of these modifications is still fragmentary. In this study, we have uncovered an intrinsic affinity between the SUMO E2 and the Cys domain of SUMO E1. NMR studies in combination with paramagnetic spin labeling demonstrate that this interaction is mediated by previously unknown interfaces on both E1 and E2 and places the two catalytic Cys residues of the two enzymes in close proximity. Site-directed mutagenesis and enzymatic assays indicate that the interaction is fundamentally important for the transfer of SUMO from E1 to E2. Results from this study suggest that the interaction between E2 and the Cys domain of E1 participates in guiding the E2's translocation to E1's enzymatic active site in ubiquitin-like modifications.

| S-EPMC1978417 | biostudies-literature

Targeting a helix-in-groove interaction between E1 and E2 blocks ubiquitin transfer.

Project description:The ubiquitin-proteasome system (UPS) is a highly regulated protein disposal process critical to cell survival. Inhibiting the pathway induces proteotoxic stress and can be an effective cancer treatment. The therapeutic window observed upon proteasomal blockade has motivated multiple UPS-targeting strategies, including preventing ubiquitination altogether. E1 initiates the cascade by transferring ubiquitin to E2 enzymes. A small molecule that engages the E1 ATP-binding site and derivatizes ubiquitin disrupts enzymatic activity and kills cancer cells. However, binding-site mutations cause resistance, motivating alternative approaches to block this promising target. We identified an interaction between the E2 N-terminal alpha-1 helix and a pocket within the E1 ubiquitin-fold domain as a potentially druggable site. Stapled peptides modeled after the E2 alpha-1 helix bound to the E1 groove, induced a consequential conformational change and inhibited E1 ubiquitin thiotransfer, disrupting E2 ubiquitin charging and ubiquitination of cellular proteins. Thus, we provide a blueprint for a distinct E1-targeting strategy to treat cancer.

| S-EPMC7904387 | biostudies-literature

A unique E1-E2 interaction required for optimal conjugation of the ubiquitin-like protein NEDD8.

Project description:Ubiquitin-like proteins (UBLs) such as NEDD8 are transferred to their targets by distinct, parallel, multienzyme cascades that involve the sequential action of E1, E2 and E3 enzymes. How do enzymes within a particular UBL conjugation cascade interact with each other? We report here that the unique N-terminal sequence of NEDD8's E2, Ubc12, selectively recruits NEDD8's E1 to promote thioester formation between Ubc12 and NEDD8. A peptide corresponding to Ubc12's N terminus (Ubc12N26) specifically binds and inhibits NEDD8's E1, the heterodimeric APPBP1-UBA3 complex. The structure of APPBP1-UBA3- Ubc12N26 reveals conserved Ubc12 residues docking in a groove generated by loops conserved in UBA3s but not other E1s. These data explain why the Ubc12-UBA3 interaction is unique to the NEDD8 pathway. These studies define a novel mechanism for E1-E2 interaction and show how enzymes within a particular UBL conjugation cascade can be tethered together by unique protein-protein interactions emanating from their common structural scaffolds.

| S-EPMC2862556 | biostudies-literature

E1-E2 interactions in ubiquitin and Nedd8 ligation pathways.

Project description:Initial rates of E1-catalyzed E2 transthiolation have been used as a reporter function to probe the mechanism of 125I-ubiquitin transfer between activation and ligation half-reactions of ubiquitin conjugation. A functional survey of 11 representative human E2 paralogs reveals similar Km for binding to human Uba1 ternary complex (Km(ave)=121±72 nm) and kcat for ubiquitin transfer (kcat(ave)=4.0±1.2 s(-1)), suggesting that they possess a conserved binding site and transition state geometry and that they compete for charging through differences in intracellular concentration. Sequence analysis and mutagenesis localize this binding motif to three basic residues within Helix 1 of the E2 core domain, confirmed by transthiolation kinetics. Partial conservation of the motif among E2 paralogs not recognized by Uba1 suggests that another factor(s) account for the absolute specificity of cognate E2 binding. Truncation of the Uba1 carboxyl-terminal ?-grasp domain reduces cognate Ubc2b binding by 31-fold and kcat by 3.5×10(4)-fold, indicating contributions to E2 binding and transition state stabilization. Truncation of the paralogous domain from the Nedd8 activating enzyme has negligible effect on cognate Ubc12 transthiolation but abrogates E2 specificity toward non-cognate carrier proteins. Exchange of the ?-grasp domains between ubiquitin and Nedd8 activating enzymes fails to reverse the effect of truncation. Thus, the conserved Helix 1 binding motif and the ?-grasp domain direct general E2 binding, whereas the latter additionally serves as a specificity filter to exclude charging of non-cognate E2 paralogs in order to maintain the fidelity of downstream signaling.

| S-EPMC3249083 | biostudies-literature

Two classes of human papillomavirus type 16 E1 mutants suggest pleiotropic conformational constraints affecting E1 multimerization, E2 interaction, and interaction with cellular proteins.

Project description:Random mutagenesis of human papillomavirus type 16 (HPV16) E1 was used to generate E1 missense mutants defective for interaction with either hUBC9 or 16E1-BP, two cDNAs encoding proteins that have been identified by their ability to interact with HPV16 E1 in two-hybrid assays. hUBC9, the human counterpart of Saccharomyces cerevisiae UBC9, is a ubiquitin-conjugating enzyme known to be involved in cell cycle progression. 16E1-BP encodes a protein of no known function but does contain an ATPase signature motif. Eight hUBC9 or 16E1-BP interaction-defective HPV16 E1 missense mutants were identified and characterized for origin-dependent transient DNA replication, ATPase activity, and various protein-protein interaction phenotypes. Six of these mutant E1 proteins were significantly impaired for replication. Among these, two classes of replication-defective HPV16 E1 missense mutants were observed. One class, represented by the S330R replication-defective mutant (containing an S-to-R change at position 330), remained competent for all protein-protein interactions tested, with the exception of hUBC9 association. Furthermore, this mutant, unlike the other replication-defective HPV16 E1 missense mutants, had a strong dominant negative replication phenotype in transient-replication assays. The other class, represented by five of the missense mutants, was defective for multiple protein-protein interactions, usually including, but not limited to, the interaction defect for which each mutant was originally selected. In many cases, a single missense mutation in one region of HPV16 E1 had pleiotropic effects, even upon activities thought to be associated with other domains of HPV16 E1. This suggests that E1 proteins are not modular but may instead be composed of multiple structurally and/or functionally interdependent domains.

| S-EPMC191850 | biostudies-other

Ubiquitin-Mimicking Peptides Transfer Differentiates by E1 and E2 Enzymes.

Project description:Ubiquitin and ubiquitin like proteins (UBLs) play key roles in eukaryotes. These proteins are attached to their target proteins through an E1-E2-E3 cascade and modify the functions of these proteins. Since the discovery of ubiquitin, several UBLs have been identified, including Nedd8, SUMO, ISG15, and Atg8. Ubiquitin and UBLs share a similar three-dimensional structure: ?-grasp fold and an X-X-[R/A/E/K]-X-X-[G/X]-G motif at the C-terminus. We have previously reported that ubiquitin, Nedd8, and SUMO mimicking peptides which all contain the conserved motif X-X-[R/A/E/K]-X-X-[G/X]-G still retained their reactivity toward their corresponding E1, E2, and E3 enzymes. In our current study, we investigated whether such C-terminal peptides could still be transferred onto related pathway enzymes to probe the function of these enzymes when they are fused with a protein. By bioinformatic search of protein databases, we selected eight proteins carrying the X-X-[R/A/E/K]-X-X-[G/X]-G motif at the C-terminus of the ?-grasp fold. We synthesized the C-terminal sequences of these candidates as short peptides and found that three of them showed significant reactivity with the ubiquitin E1 enzyme Ube1. We next fused the three reactive short peptides to three different protein frames, including their respective native protein frames, a ubiquitin frame and a peptidyl carrier protein (PCP) frame, and measured the reactivities of these peptide-fused proteins with Ube1. Peptide-fused proteins on ubiquitin and PCP frames showed obvious reactivity with Ube1. However, when we measured E2 UbcH7 transfer, we found that the PCP-peptide fusions lost their reactivity with UbcH7. Taken together, these results suggested that the recognition of E2 enzymes with peptide-fused proteins depended not only on the C-terminal sequences of the ubiquitin-mimicking peptides, but also on the overall structures of the protein frames.

| S-EPMC6136576 | biostudies-literature

Orthogonal ubiquitin transfer through engineered E1-E2 cascades for protein ubiquitination.

Project description:Protein modification by ubiquitin (UB) controls diverse cellular processes. UB is conjugated to cellular proteins by sequential transfer through an E1-E2-E3 enzymatic cascade. The cross-activities of 2 E1s, 50 E2s and thousands of E3s encoded by the human genome make it difficult to identify the substrate proteins of a specific E3 enzyme in the cell. One way to solve this problem is to engineer an orthogonal UB transfer (OUT) cascade in which the engineered UB (xUB) is relayed by engineered E1, E2 and E3 enzymes (xE1, xE2, xE3) to modify the substrate proteins of a specific E3. Here, we use phage display and mutagenesis to construct xUB-xE1 and xE1-xE2 pairs that are orthogonal to the native E1 and E2 enzymes. Our work on engineering the UB transfer cascades will enable us to use OUT to map the signal transduction networks mediated by protein ubiquitination.

| S-EPMC4104569 | biostudies-literature

A cascading activity-based probe sequentially targets E1-E2-E3 ubiquitin enzymes.

Project description:Post-translational modifications of proteins with ubiquitin (Ub) and ubiquitin-like modifiers (Ubls), orchestrated by a cascade of specialized E1, E2 and E3 enzymes, control a wide range of cellular processes. To monitor catalysis along these complex reaction pathways, we developed a cascading activity-based probe, UbDha. Similarly to the native Ub, upon ATP-dependent activation by the E1, UbDha can travel downstream to the E2 (and subsequently E3) enzymes through sequential trans-thioesterifications. Unlike the native Ub, at each step along the cascade, UbDha has the option to react irreversibly with active site cysteine residues of target enzymes, thus enabling their detection. We show that our cascading probe 'hops' and 'traps' catalytically active Ub-modifying enzymes (but not their substrates) by a mechanism diversifiable to Ubls. Our founder methodology, amenable to structural studies, proteome-wide profiling and monitoring of enzymatic activity in living cells, presents novel and versatile tools to interrogate Ub and Ubl cascades.

| S-EPMC5108872 | biostudies-literature

Basis for a ubiquitin-like protein thioester switch toggling E1-E2 affinity.

Project description:Ubiquitin-like proteins (UBLs) are conjugated by dynamic E1-E2-E3 enzyme cascades. E1 enzymes activate UBLs by catalysing UBL carboxy-terminal adenylation, forming a covalent E1 throught UBL thioester intermediate, and generating a thioester-linked E2 throught UBL product, which must be released for subsequent reactions. Here we report the structural analysis of a trapped UBL activation complex for the human NEDD8 pathway, containing NEDD8's heterodimeric E1 (APPBP1-UBA3), two NEDD8s (one thioester-linked to E1, one noncovalently associated for adenylation), a catalytically inactive E2 (Ubc12), and MgATP. The results suggest that a thioester switch toggles E1-E2 affinities. Two E2 binding sites depend on NEDD8 being thioester-linked to E1. One is unmasked by a striking E1 conformational change. The other comes directly from the thioester-bound NEDD8. After NEDD8 transfer to E2, reversion to an alternate E1 conformation would facilitate release of the E2 throught NEDD8 thioester product. Thus, transferring the UBL's thioester linkage between successive conjugation enzymes can induce conformational changes and alter interaction networks to drive consecutive steps in UBL cascades.

| S-EPMC2821831 | biostudies-literature

OmicsDI is part of the ELIXIR infrastructure

OmicsDI is an Elixir interoperability service. Learn more ›

OmicsDI Databases

PRIDE
PeptideAtlas
MassIVE
JPOST Repository
Physiome Model Repository

EGA
EVA
ENA
LINCS
PAXDB
Cell Collective

MetaboLights
Metabolomics Workbench
MetabolomeExpress
GNPS
BioModels
FAIRDOMHub

ArrayExpress
dbGaP
ExpressionAtlas
GEO
NODE

Information

Databases
Help
API
Contact us
Code on GitHub
Terms of use
Submit Data