Structure of a conjugating enzyme-ubiquitin thiolester intermediate reveals a novel role for the ubiquitin tail

BACKGROUND: Ubiquitin-conjugating enzymes (E2s) are central enzymes involved in ubiquitin-mediated protein degradation. During this process, ubiquitin (Ub) and the E2 protein form an unstable E2-Ub thiolester intermediate prior to the transfer of ubiquitin to an E3-ligase protein and the labeling of a substrate for degradation. A series of complex interactions occur among the target substrate, ubiquitin, E2, and E3 in order to efficiently facilitate the transfer of the ubiquitin molecule. However, due to the inherent instability of the E2-Ub thiolester, the structural details of this complex intermediate are not known. RESULTS: A three-dimensional model of the E2-Ub thiolester intermediate has been determined for the catalytic domain of the E2 protein Ubc1 (Ubc1(Delta450)) and ubiquitin from S. cerevisiae. The interface of the E2-Ub intermediate was determined by kinetically monitoring thiolester formation by 1H-(15)N HSQC spectra by using combinations of 15N-labeled and unlabeled Ubc1(Delta450) and Ub proteins. By using the surface interface as a guide and the X-ray structures of Ub and the 1.9 A structure of Ubc1(Delta450) determined here, docking simulations followed by energy minimization were used to produce the first model of a E2-Ub thiolester intermediate. CONCLUSIONS: Complementary surfaces were found on the E2 and Ub proteins whereby the C terminus of Ub wraps around the E2 protein terminating in the thiolester between C88 (Ubc1(Delta450)) and G76 (Ub). The model supports in vivo and in vitro experiments of E2 derivatives carrying surface residue substitutions. Furthermore, the model provides insights into the arrangement of Ub, E2, and E3 within a ternary targeting complex.     

E2-binding surface on Uba3 β-grasp domain undergoes a conformational transition

The covalent attachment of ubiquitin (Ub) and ubiquitin‐like (Ubl) proteins to various eukaryotic targets plays critical roles in regulating numerous cellular processes. E1‐activating enzymes are critical, because they catalyze activation of their cognate Ub/Ubl protein and are responsible for its transfer to the correct E2‐conjugating enzyme(s). The activating enzyme for neural‐precursor‐cell‐expressed developmentally downregulated 8 (NEDD8) is a heterodimer composed of APPBP1 and Uba3 subunits. The carboxyl terminal ubiquitin‐like β‐grasp domain of human Uba3 (Uba3‐βGD) has been suggested as a key E2‐binding site defining E2 specificity. In crystal structures of free E1 and the NEDD8‐E1 complex, the E2‐binding surface on the domain was missing from the electron density. However, when complexed with various E2s, this missing segment adopts a kinked α‐helix. Here, we demonstrate that Uba3‐βGD is an independently folded domain in solution and that residues involved in E2 binding are absent from the NMR spectrum, indicating that the E2‐binding surface on Uba3‐βGD interconverts between multiple conformations, analogous to a similar conformational transition observed in the E2‐binding surface of SUMO E1. These results suggest that access to multiple conformational substates is an important feature of the E1–E2 interaction. Proteins 2012;. © 2012 Wiley Periodicals, Inc.     

E2 conjugating enzyme selectivity and requirements for function of the E3 ubiquitin ligase CHIP

The transfer of ubiquitin (Ub) to a substrate protein requires a cascade of E1 activating, E2 conjugating, and E3 ligating enzymes. E3 Ub ligases containing U-box and RING domains bind both E2～Ub conjugates and substrates to facilitate transfer of the Ub molecule. Although the overall mode of action of E3 ligases is well established, many of the mechanistic details that determine the outcome of ubiquitination are poorly understood. CHIP (carboxyl terminus of Hsc70-interacting protein) is a U-box E3 ligase that serves as a co-chaperone to heat shock proteins and is critical for the regulation of unfolded proteins in the cytosol. We have performed a systematic analysis of the interactions of CHIP with E2 conjugating enzymes and found that only a subset bind and function. Moreover, some E2 enzymes function in pairs to create products that neither create individually. Characterization of the products of these reactions showed that different E2 enzymes produce different ubiquitination products, i.e. that E2 determines the outcome of Ub transfer. Site-directed mutagenesis on the E2 enzymes Ube2D1 and Ube2L3 (UbcH5a and UbcH7) established that an SPA motif in loop 7 of E2 is required for binding to CHIP but is not sufficient for activation of the E2～Ub conjugate and consequent ubiquitination activity. These data support the proposal that the E2 SPA motif provides specificity for binding to CHIP, whereas activation of the E2～Ub conjugate is derived from other molecular determinants.     

Molecular and structural insight into lysine selection on substrate and ubiquitin lysine 48 by the ubiquitin-conjugating enzyme Cdc34

The attachment of ubiquitin (Ub) to lysines on substrates or itself by ubiquitin-conjugating (E2) and ubiquitin ligase (E3) enzymes results in protein ubiquitination. Lysine selection is important for generating diverse substrate-Ub structures and targeting proteins to different fates; however, the mechanisms of lysine selection are not clearly understood. The positioning of lysine(s) toward the E2/E3 active site and residues proximal to lysines are critical in their selection. We investigated determinants of lysine specificity of the ubiquitin-conjugating enzyme Cdc34, toward substrate and Ub lysines. Evaluation of the relative importance of different residues positioned −2, −1, +1 and +2 toward ubiquitination of its substrate, Sic1, on lysine 50 showed that charged residues in the −1 and −2 positions negatively impact on ubiquitination. Modeling suggests that charged residues at these positions alter the native salt-bridge interactions in Ub and Cdc34, resulting in misplacement of Sic1 lysine 50 in the Cdc34 catalytic cleft. During polyubiquitination, Cdc34 showed a strong preference for Ub lysine 48 (K48), with lower activity towards lysine 11 (K11) and lysine 63 (K63). Mutating the −2, −1, +1 and +2 sites surrounding K11 and K63 to mimic those surrounding K48 did not improve their ubiquitination, indicating that further determinants are important for Ub K48 specificity. Modeling the ternary structure of acceptor Ub with the Cdc34~Ub complex as well as in vitro ubiquitination assays unveiled the importance of K6 and Q62 of acceptor Ub for Ub K48 polyubiquitination. These findings provide molecular and structural insight into substrate lysine and Ub K48 specificity by Cdc34.     

The basis for selective E1-E2 interactions in the ISG15 conjugation system

E1 and E2 enzymes coordinate the first steps in conjugation of ubiquitin (Ub) and ubiquitin-like proteins (Ubls). ISG15 is an interferon-alpha/beta-induced Ubl, and the E1 and E2 enzymes for ISG15 conjugation are Ube1L and UbcH8, respectively. UbcH7 is the most closely related E2 to UbcH8, yet it does not function in ISG15 conjugation in vivo, while both UbcH7 and UbcH8 have been reported to function in Ub conjugation. Kinetic analyses of wild-type and chimeric E2s were performed to determine the basis for preferential activation of UbcH8 by Ube1L and to determine whether UbcH8 is activated equally well by Ube1L and E1(Ub) (Ube1). K(m) determinations confirmed the strong preference of Ube1L for UbcH8 over UbcH7 (a 29-fold K(m) difference), similar to the preference of E1(Ub) for UbcH7 over UbcH8 (a 36-fold K(m) difference). Thioester assays of chimeric E2s identified two structural elements within residues 1-39 of UbcH8 that play a major role in defining Ube1L-UbcH8 specificity: the alpha1-helix and the beta1-beta2 region. The C-terminal ubiquitin fold domain (UFD) of Ube1L was required for transfer of ISG15 to UbcH8 and for binding of Ube1L to UbcH8. Replacement of the Ube1L UFD with that from E1(Ub) resulted in preferential transfer of ISG15 to UbcH7. Together, these results indicate that Ube1L discriminates between UbcH8 and closely related Ub E2s based on specific interactions between the Ube1L UFD and determinants within the N-terminal region of UbcH8.     

A novel class of herpesvirus-encoded membrane-bound E3 ubiquitin ligases regulates endocytosis of proteins involved in immune recognition.

Kaposi's sarcoma-associated herpesvirus encodes two transmembrane proteins (modulator of immune recognition [MIR]1 and MIR2) that downregulate cell surface molecules (MHC-I, B7.2, and ICAM-1) involved in the immune recognition of infected cells. This downregulation results from enhanced endocytosis and subsequent endolysosomal degradation of the target proteins. Here, we show that expression of MIR1 and MIR2 leads to ubiquitination of the cytosolic tail of their target proteins and that ubiquitination is essential for their removal from the cell surface. MIR1 and MIR2 both contain cytosolic zinc fingers of the PHD subfamily, and these structures are required for this activity. In vitro, addition of a MIR2-glutathione S-transferase (GST) fusion protein to purified E1 and E2 enzymes leads to transfer of ubiquitin (Ub) to GST-containing targets in an ATP- and E2-dependent fashion; this reaction is abolished by mutation of the Zn-coordinating residues of the PHD domain. Thus, MIR2 defines a novel class of membrane-bound E3 Ub ligases that modulates the trafficking of host cell membrane proteins.     

Structure and functional interactions of the Tsg101 UEV domain

Human Tsg101 plays key roles in HIV budding and in cellular vacuolar protein sorting (VPS). In performing these functions, Tsg101 binds both ubiquitin (Ub) and the PTAP tetrapeptide 'late domain' motif located within the viral Gag protein. These interactions are mediated by the N-terminal domain of Tsg101, which belongs to the catalytically inactive ubiquitin E2 variant (UEV) family. We now report the structure of Tsg101 UEV and chemical shift mapping of the Ub and PTAP binding sites. Tsg101 UEV resembles canonical E2 ubiquitin conjugating enzymes, but has an additional N-terminal helix, an extended beta-hairpin that links strands 1 and 2, and lacks the two C-terminal helices normally found in E2 enzymes. PTAP-containing peptides bind in a hydrophobic cleft exposed by the absence of the C-terminal helices, whereas ubiquitin binds in a novel site surrounding the beta-hairpin. These studies provide a structural framework for understanding how Tsg101 mediates the protein-protein interactions required for HIV budding and VPS.     

Ubiquitin Ser65 phosphorylation affects ubiquitin structure,chain assembly and hydrolysis

The protein kinase PINK1 was recently shown to phosphorylate ubiquitin (Ub) on Ser65, and phosphoUb activates the E3 ligase Parkin allosterically. Here, we show that PINK1 can phosphorylate every Ub in Ub chains. Moreover, Ser65 phosphorylation alters Ub structure, generating two conformations in solution. A crystal structure of the major conformation resembles Ub but has altered surface properties. NMR reveals a second phosphoUb conformation in which β5-strand slippage retracts the C-terminal tail by two residues into the Ub core. We further show that phosphoUb has no effect on E1-mediated E2 charging but can affect discharging of E2 enzymes to form polyUb chains. Notably, UBE2R1- (CDC34), UBE2N/UBE2V1- (UBC13/UEV1A), TRAF6- and HOIP-mediated chain assembly is inhibited by phosphoUb. While Lys63-linked poly-phosphoUb is recognized by the TAB2 NZF Ub binding domain (UBD), 10 out of 12 deubiquitinases (DUBs), including USP8, USP15 and USP30, are impaired in hydrolyzing phosphoUb chains. Hence, Ub phosphorylation has repercussions for ubiquitination and deubiquitination cascades beyond Parkin activation and may provide an independent layer of regulation in the Ub system.     

Regulating the Regulators: Recent Revelations in the Control of E3 Ubiquitin Ligases

Since its discovery as a post-translational signal for protein degradation, our understanding of ubiquitin (Ub) has vastly evolved. Today, we recognize that the role of Ub signaling is expansive and encompasses diverse processes including cell division, the DNA damage response, cellular immune signaling, and even organismal development. With such a wide range of functions comes a wide range of regulatory mechanisms that control the activity of the ubiquitylation machinery. Ub attachment to substrates occurs through the sequential action of three classes of enzymes, E1s, E2s, and E3s. In humans, there are 2 E1s, ∼35 E2s, and hundreds of E3s that work to attach Ub to thousands of cellular substrates. Regulation of ubiquitylation can occur at each stage of the stepwise Ub transfer process, and substrates can also impact their own modification. Recent studies have revealed elegant mechanisms that have evolved to control the activity of the enzymes involved. In this minireview, we highlight recent discoveries that define some of the various mechanisms by which the activities of E3-Ub ligases are regulated.     

Crystal structure of the cyclin-specific ubiquitin-conjugating enzyme from clam, E2-C, at 2.0 A resolution.

The destruction of the cyclin B protein is necessary for the cell to exit from mitosis. The destruction of cyclin B occurs via the ubiquitin/proteasome system and involves a specific ubiquitin-conjugating enzyme (Ubc) that donates ubiquitin to cyclin B. Here we present the crystal structure of the cyclin-specific Ubc from clam, E2-C, determined at 2.0 A resolution. The E2-C enzyme contains an N-terminal extension in addition to the Ubc core domain. The N-terminal extension is disordered, perhaps reflecting a need for flexibility as it interacts with various partners in the ubiquitination system. The overall structure of the E2-C core domain is quite similar to those in previously determined Ubc proteins. The interaction between particular pairs of E2-C proteins in the crystal has some of the hallmarks of a functional dimer, though solution studies suggest that the E2-C protein exists as a monomer. Comparison of the E2-C structure with that of the other available Ubc structures indicates conserved surface residues that may interact with common components of the ubiquitination system. Such comparison also reveals a remarkable spine of conserved hydrophobic residues in the center of the protein that may drive the protein to fold and stabilize the protein once folded. Comparison of residues conserved only among E2-C and its homologues indicates surface areas that may be involved in mitotic-specific ubiquitination.     

Role of a non-canonical surface of Rad6 in ubiquitin conjugating activity

Rad6 is a yeast E2 ubiquitin conjugating enzyme that monoubiquitinates histone H2B in conjunction with the E3, Bre1, but can non-specifically modify histones on its own. We determined the crystal structure of a Rad6∼Ub thioester mimic, which revealed a network of interactions in the crystal in which the ubiquitin in one conjugate contacts Rad6 in another. The region of Rad6 contacted is located on the distal face of Rad6 opposite the active site, but differs from the canonical E2 backside that mediates free ubiquitin binding and polyubiquitination activity in other E2 enzymes. We find that free ubiquitin interacts weakly with both non-canonical and canonical backside residues of Rad6 and that mutations of non-canonical residues have deleterious effects on Rad6 activity comparable to those observed to mutations in the canonical E2 backside. The effect of non-canonical backside mutations is similar in the presence and absence of Bre1, indicating that contacts with non-canonical backside residues govern the intrinsic activity of Rad6. Our findings shed light on the determinants of intrinsic Rad6 activity and reveal new ways in which contacts with an E2 backside can regulate ubiquitin conjugating activity.     

Ubiquitin in motion: structural studies of the ubiquitin-conjugating enzyme∼ubiquitin conjugate

Ubiquitination of proteins provides a powerful and versatile post-translational signal in the eukaryotic cell. The formation of a thioester bond between ubiquitin (Ub) and the active site of a ubiquitin-conjugating enzyme (E2) is critical for the transfer of Ub to substrates. Assembly of a functional ubiquitin ligase (E3) complex poised for Ub transfer involves recognition and binding of an E2～Ub conjugate. Therefore, full characterization of the structure and dynamics of E2～Ub conjugates is required for further mechanistic understanding of Ub transfer reactions. Here we present characterization of the dynamic behavior of E2～Ub conjugates of two human enzymes, UbcH5c～Ub and Ubc13～Ub, in solution as determined by nuclear magnetic resonance and small-angle X-ray scattering. Within each conjugate, Ub retains great flexibility with respect to the E2, indicative of highly dynamic species that adopt manifold orientations. The population distribution of Ub conformations is dictated by the identity of the E2: the UbcH5c～Ub conjugate populates an array of extended conformations, and the population of Ubc13～Ub conjugates favors a closed conformation in which the hydrophobic surface of Ub faces helix 2 of Ubc13. We propose that the varied conformations adopted by Ub represent available binding modes of the E2～Ub species and thus provide insight into the diverse E2～Ub protein interactome, particularly with regard to interaction with Ub ligases.     

A bioluminescent assay for monitoring conjugation of ubiquitin and ubiquitin-like proteins

Post-translational modification of target proteins by ubiquitin (Ub) and ubiquitin-like (Ubl) proteins is a critical mechanism for regulating protein functions affecting diverse cellular processes. Ub/Ubl proteins are conjugated to lysine residues in substrate proteins through an adenosine triphosphate (ATP)-dependent enzymatic cascade involving enzyme 1 (E1)-activating enzyme, E2-conjugating enzyme, and E3 ligase. The amount of adenosine monophosphate (AMP) produced in the first step, involving E1-mediated Ub/Ubl activation, represents an accurate measure of Ub/Ubl transfer during the process. Here we describe a novel bioluminescent assay platform, AMP-Glo, to quantify Ub/Ubl conjugation by measuring the AMP generated. The AMP-Glo assay is performed in a two-step reaction. The first step terminates the ubiquitination reaction, depletes the remaining ATP, and converts the AMP generated in the ubiquitination reaction to adenosine diphosphate (ADP), and in the second step the ADP generated is converted to ATP, which is detected as a bioluminescent signal using luciferase/luciferin, proportional to the AMP concentration and correlated with the Ub/Ubl transfer activity. We demonstrate the use of the assay to study Ub/Ubl conjugation and screen for chemical modulators of enzymes involved in the process. Because there is a sequential enhancement in light output in the presence of E1, E2, and E3, the AMP-Glo system can be used to deconvolute inhibitor specificity.     

E2-c-Cbl recognition is necessary but not sufficient for ubiquitination activity

The E2 ubiquitin-conjugating enzymes UbcH7 and UbcH5B both show specific binding to the RING (really interesting new gene) domain of the E3 ubiquitin-protein ligase c-Cbl, but UbcH7 hardly supports ubiquitination of c-Cbl and substrate in a reconstituted system. Here, we found that neither structural changes nor subtle differences in the E2-E3 interaction surface are possible explanations for the functional specificity of UbcH5B and UbcH7 in their interaction with c-Cbl. The quick transfer of ubiquitin from the UbcH5B∼Ub thioester to c-Cbl or other ubiquitin acceptors suggests that UbcH5B might functionally be a relatively pliable E2 enzyme. In contrast, the UbcH7∼Ub thioester is too stable to transfer ubiquitin under our assay conditions, indicating that UbcH7 might be a more specific E2 enzyme. Our results imply that the interaction specificity between c-Cbl and E2 is required but not sufficient for transfer of ubiquitin to potential targets.     

Characterization of the binding interface between ubiquitin and class I human ubiquitin-conjugating enzyme 2b by multidimensional heteronuclear NMR spectroscopy in solution.

Ubiquitin-conjugating enzymes (Ubc) are involved in ubiquitination of proteins in the protein degradation pathway of eukaryotic cells. Ubc transfers the ubiquitin (Ub) molecules to target proteins by forming a thioester bond between their active site cysteine residue and the C-terminal glycine residue of ubiquitin. Here, we report on the NMR assignment and secondary structure of class I human ubiquitin-conjugating enzyme 2b (HsUbc2b). Chemical shift perturbation studies allowed us to map the contact area and binding interface between ubiquitin and HsUbc2b by1H-15N HSQC NMR spectroscopy. The serine mutant of the active site Cys88 of HsUbc2b was employed to obtain a relatively stable covalent ubiquitin complex of HsUbc2b(C88S). Changes in chemical shifts of amide protons and nitrogen atoms induced by the formation of the covalent complex were measured by preparing two segmentally labeled complexes with either ubiquitin or HsUbc2b(C88S)15N-labeled. In ubiquitin, the interaction is primarily sensed by the C-terminal segment Val70 - Gly76, and residues Lys48 and Gln49. The surface area on ubiquitin, as defined by these residues, overlaps partially with the presumed binding site with ubiquitin-activating enzyme (E1). In HsUbc2b, most of the affected residues cluster in the vicinity of the active site, namely, around the active site Cys88 itself, the second alpha-helix, and the flexible loop which connects helices alpha2 and alpha3 and which is adjacent to the active site. An additional site on HsUbc2b for a weak interaction with ubiquitin could be detected in a titration study where the two proteins were not covalently linked. This site is located on the backside of HsUbc2b opposite to the active site and is part of the beta-sheet. The covalent and non-covalent interaction sites are clearly separated on the HsUbc2b surface, while no such clear-cut segregation of the interaction area was observed on ubiquitin.     

E2 enzymes: more than just middle men

Ubiquitin-conjugating enzymes (E2s) are the central players in the trio of enzymes responsible for the attachment of ubiquitin (Ub) to cellular proteins. Humans have ∼40 E2s that are involved in the transfer of Ub or Ub-like (Ubl) proteins (e.g., SUMO and NEDD8). Although the majority of E2s are only twice the size of Ub, this remarkable family of enzymes performs a variety of functional roles. In this review, we summarize common functional and structural features that define unifying themes among E2s and highlight emerging concepts in the mechanism and regulation of E2s.     

Certain pairs of ubiquitin-conjugating enzymes (E2s) and ubiquitin-protein ligases (E3s) synthesize nondegradable forked ubiquitin chains containing all possible isopeptide linkages

It is generally assumed that a specific ubiquitin ligase (E3) links protein substrates to polyubiquitin chains containing a single type of isopeptide linkage, and that chains composed of linkages through Lys(48), but not through Lys(63), target proteins for proteasomal degradation. However, when we carried out a systematic analysis of the types of ubiquitin (Ub) chains formed by different purified E3s and Ub-conjugating enzymes (E2s), we found, using Ub mutants and mass spectrometry, that the U-box E3, CHIP, and Ring finger E3s, MuRF1 and Mdm2, with the E2, UbcH5, form a novel type of Ub chain that contains all seven possible linkages, but predominantly Lys(48), Lys(63), and Lys(11) linkages. Also, these heterogeneous chains contain forks (bifurcations), where two Ub molecules are linked to the adjacent lysines at Lys(6) + Lys(11), Lys(27) + Lys(29), or Lys(29) + Lys(33) on the preceding Ub molecule. However, the HECT domain E3s, E6AP and Nedd4, with the same E2, UbcH5, form homogeneous chains exclusively, either Lys(48) chains (E6AP) or Lys(63) chains (Nedd4). Furthermore, with other families of E2s, CHIP and MuRF1 synthesize homogeneous Ub chains on the substrates. Using the dimeric E2, UbcH13/Uev1a, they attach Lys(63) chains, but with UbcH1 (E2-25K), MuRF1 synthesizes Lys(48) chains on the substrate. We then compared the capacity of the forked heterogeneous chains and homogeneous chains to support proteasomal degradation. When troponin I was linked by MuRF1 to a Lys(48)-Ub chain or, surprisingly, to a Lys(63)-Ub chain, troponin I was degraded rapidly by pure 26S proteasomes. However, when linked to the mixed forked chains, troponin I was degraded quite poorly, and its polyUb chain, especially the forked linkages, was disassembled slowly by proteasome-associated isopeptidases. Because these Ring finger and U-box E3s with UbcH5 target proteins for degradation in vivo, but Lys(63) chains do not, cells probably contain additional factors that prevent formation of such nondegradable Ub-conjugates and that protect proteins linked to Lys(63)-Ub chains from proteasomal degradation.     

Synthetic biology approach to reconstituting the ubiquitylation cascade in bacteria

Covalent modification of proteins with ubiquitin (Ub) is widely implicated in the control of protein function and fate. Over 100 deubiquitylating enzymes rapidly reverse this modification, posing challenges to the biochemical and biophysical characterization of ubiquitylated proteins. We circumvented this limitation with a synthetic biology approach of reconstructing the entire eukaryotic Ub cascade in bacteria. Co‐expression of affinity‐tagged substrates and Ub with E1, E2 and E3 enzymes allows efficient purification of ubiquitylated proteins in milligram quantity. Contrary to in‐vitro assays that lead to spurious modification of several lysine residues of Rpn10 (regulatory proteasomal non‐ATPase subunit), the reconstituted system faithfully recapitulates its monoubiquitylation on lysine 84 that is observed in vivo. Mass spectrometry revealed the ubiquitylation sites on the Mind bomb E3 ligase and the Ub receptors Rpn10 and Vps9. Förster resonance energy transfer (FRET) analyses of ubiquitylated Vps9 purified from bacteria revealed that although ubiquitylation occurs on the Vps9‐GEF domain, it does not affect the guanine nucleotide exchanging factor (GEF) activity in vitro. Finally, we demonstrated that ubiquitylated Vps9 assumes a closed structure, which blocks additional Ub binding. Characterization of several ubiquitylated proteins demonstrated the integrity, specificity and fidelity of the system, and revealed new biological findings.