Ubiquitin chain conformation regulates recognition and activity of interacting proteins (original) (raw)
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Mechanism of Polyubiquitin Chain Recognition by the Human Ubiquitin Conjugating Enzyme Ube2g2
Journal of Biological Chemistry, 2011
Ube2g2 is a human ubiquitin conjugating (E2) enzyme involved in the endoplasmic reticulum-associated degradation pathway, which is responsible for the identification and degradation of unfolded and misfolded proteins in the endoplasmic reticulum compartment. The Ube2g2-specific role is the assembly of Lys-48-linked polyubiquitin chains, which constitutes a signal for proteasomal degradation when attached to a substrate protein. NMR chemical shift perturbation and paramagnetic relaxation enhancement approaches were employed to characterize the binding interaction between Ube2g2 and ubiquitin, Lys-48-linked diubiquitin, and Lys-63-linked diubiquitin. Results demonstrate that ubiquitin binds to Ube2g2 with an affinity of 90 M in two different orientations that are rotated by 180°in models generated by the RosettaDock modeling suite. The binding of Ube2g2 to Lys-48-and Lys-63linked diubiquitin is primarily driven by interactions with individual ubiquitin subunits, with a clear preference for the subunit containing the free Lys-48 or Lys-63 side chain (i.e. the distal subunit). This preference is particularly striking in the case of Lys-48-linked diubiquitin, which exhibits an ϳ3-fold difference in affinities between the two ubiquitin subunits. This difference can be attributed to the partial steric occlusion of the subunit whose Lys-48 side chain is involved in the isopeptide linkage. As such, these results suggest that Lys-48linked polyubiquitin chains may be designed to bind certain proteins like Ube2g2 such that the terminal ubiquitin subunit carrying the reactive Lys-48 side chain can be positioned properly for chain elongation regardless of chain length.
Molecular discrimination of structurally equivalent Lys 63‐linked and linear polyubiquitin chains
EMBO reports, 2009
At least eight types of ubiquitin chain exist, and individual linkages affect distinct cellular processes. The only distinguishing feature of differently linked ubiquitin chains is their structure, as polymers of the same unit are chemically identical. Here, we have crystallized Lys 63-linked and linear ubiquitin dimers, revealing that both adopt equivalent open conformations, forming no contacts between ubiquitin molecules and thereby differing significantly from Lys 48-linked ubiquitin chains. We also examined the specificity of various deubiquitinases (DUBs) and ubiquitin-binding domains (UBDs). All analysed DUBs, except CYLD, cleave linear chains less efficiently compared with other chain types, or not at all. Likewise, UBDs can show chain specificity, and are able to select distinct linkages from a ubiquitin chain mixture. We found that the UBAN (ubiquitin binding in ABIN and NEMO) motif of NEMO (NF-jB essential modifier) binds to linear chains exclusively, whereas the NZF (Npl4 zinc finger) domain of TAB2 (TAK1 binding protein 2) is Lys 63 specific. Our results highlight remarkable specificity determinants within the ubiquitin system.
Nature Structural & Molecular Biology, 2010
Ubiquitin is a versatile cellular signaling molecule that can form polymers of eight different linkages, and individual linkage-types have been associated with distinct cellular functions. Though little is currently known about Lys11-linked ubiquitin chains, recent data indicate that they may be as abundant as Lys48-linkages and involved in vital cellular processes. Here we report the generation of Lys11-linked polyubiquitin in vitro, for which the Lys11-specific E2 enzyme UBE2S was fused to a ubiquitin binding domain. Crystallography and NMR analyses of Lys11-linked diubiquitin reveal that Lys11-linked chains adopt compact conformations in which Ile44 is solvent exposed. Furthermore, we identify the OTU family deubiquitinase Cezanne as the first deubiquitinase with Lys11-linkage preference. Our data highlight the intrinsic specificity of the ubiquitin system that extends to Lys11-linked chains, and emphasize that differentially linked polyubiquitin chains must be regarded as independent posttranslational modifications.
Conformational Dynamics of Wild-type Lys-48-linked Diubiquitin in Solution
Journal of Biological Chemistry, 2011
Proteasomal degradation is mediated through modification of target proteins by Lys-48-linked polyubiquitin (polyUb) chain, which interacts with several binding partners in this pathway through hydrophobic surfaces on individual Ub units. However, the previously reported crystal structures of Lys-48-linked diUb exhibit a closed conformation with sequestered hydrophobic surfaces. NMR studies on mutated Lys-48-linked diUb indicated a pH-dependent conformational equilibrium between closed and open states with the predominance of the former under neutral conditions (90% at pH 6.8). To address the question of how Ub-binding proteins can efficiently access the sequestered hydrophobic surfaces of Ub chains, we revisited the conformational dynamics of Lys-48-linked diUb in solution using wild-type diUb and cyclic forms of diUb in which the Ub units are connected through two Lys-48-mediated isopeptide bonds. Our newly determined crystal structure of wild-type diUb showed an open conformation, whereas NMR analyses of cyclic Lys-48-linked diUb in solution revealed that its structure resembled the closed conformation observed in previous crystal structures. Comparison of a chemical shift of wild-type diUb with that of monomeric Ub and cyclic diUb, which mimic the open and closed states, respectively, with regard to the exposure of hydrophobic surfaces to the solvent indicates that wild-type Lys-48-linked diUb in solution predominantly exhibits the open conformation (75% at pH 7.0), which becomes more populated upon lowering pH. The intrinsic properties of Lys-48-linked Ub chains to adopt the open conformation may be advantageous for interacting with Ub-binding proteins. Ubiquitin (Ub) 3 is a small protein composed of 76 amino acid residues and plays regulatory roles in various cellular events, including cell cycle progression, DNA repair, transcriptional regulation, and apoptosis (1, 2). These Ub regulatory functions are expressed by its modification of target proteins through the formation of isopeptide linkages at the C terminus. Ubiquitination is catalyzed by the sequential action of the Ub-activating enzyme E1, Ub-conjugating enzyme E2, and Ub-protein ligase E3. The C-terminal group of Ub can also be linked to another Ub (termed the distal and proximal moieties, respectively) through all seven lysine residues at positions 6, 11, 27, 29, 33, 48, and 63 as well as the N terminus, giving rise to various types of polyUb chains (3-7), which mediate diverse signals determining the fate of ubiquitinated proteins. The best characterized case is the Lys-48-linked polyUb chain that serves as a tag for protein degradation by 26 S proteasomes (1). In the Ub/proteasome-mediated proteolytic pathway, Lys-48-linked polyUb is recognized by several proteins possessing Ub-binding motifs (8, 9). These include the proteasomal subunits S5a/Rpn10 and Rpn13 as well as the Ub receptors hHR23/ Rad23, Dsk2/Dph1, and Ddi1/Mud1. NMR studies reported the interactions with the ubiquitin-associated domain of hHR23A and the ubiquitin-interacting motifs of S5a using Lys-48-linked diUb as a minimal model (10, 11). These studies indicated that hydrophobic surfaces, including Val-70 in both Ub units, are involved in the interactions with these Ub-binding motifs. Conformation of Lys-48-linked diUb has also been characterized in the absence of its binding partners. The crystal structures of Lys-48-linked diUb have been solved for two different crystal forms grown under different crystallization conditions (12, 13). Crystallographic studies showed that Lys-48-linked diUb exhibits a "closed" conformation in which hydrophobic surfaces in both Ub units are packed against each other and shielded from the solvent. Identical Ub-Ub interaction modes have been observed in the crystal structures of an engineered tetraUb (14, 15) in which Lys-48 of the second and fourth Ub units are substituted by thialysine and arginine, respectively, and Gly-76 is deleted in the first Ub, and a cyclic tetraUb (16) in which the C terminus of the first Ub is conjugated to Lys-48 of the fourth Ub. Conformational characterization of Lys-48-linked diUb in solution has also been performed using NMR spectroscopy (17, 18). Fushman and co-workers reported a series of NMR studies
Structural basis for distinct roles of Lys63- and Lys48-linked polyubiquitin chains
Genes to Cells, 2004
Ubiquitination, a modification in which single or multiple ubiquitin molecules are attached to a protein, serves as a signalling function that controls a wide variety of cellular processes. To date, two major forms of polyubiquitin chain have been functionally characterized, in which the isopeptide bond linkages involve Lys48 or Lys63. Lys48-linked polyubiquitin tagging is mostly used to target proteins for degradation by the proteasome, whereas Lys63-linked polyubiquitination has been linked to numerous cellular events that do not rely on degradative signalling via the proteasome. Apparently linkage-specific conformations of polyubiquitin chains are important for these cellular functions, but the structural bases distinguishing Lys48-and Lys63-linked chains remain elusive.
Assembly and structure of Lys33-linked polyubiquitin reveals distinct conformations
The Biochemical journal, 2015
Ubiquitylation regulates a multitude of biological processes and this versatility stems from the ability of ubiquitin to form topologically different polymers of eight different linkage types. While some linkages have been studied in detail, other linkage types including Lys33-linked polyubiquitin are poorly understood. Here we identify an enzymatic system for the large-scale assembly of Lys33 chains by combining the HECT (homologous to the E6-AP carboxyl terminus) E3 ligase AREL1 (apoptosis-resistant E3 ubiquitin protein ligase 1) with linkage selective deubiquitinases (DUBs). Moreover, this first characterisation of the chain selectivity of AREL1 indicates its preference for assembling Lys33- and Lys11-linked ubiquitin chains. Intriguingly, the crystal structure of Lys33-linked diubiquitin (diUb) reveals that it adopts a compact conformation very similar to that observed for Lys11-linked diUb. In contrast, crystallographic analysis of Lys33-linked triUb reveals a more extended con...
Noncovalent Dimerization of Ubiquitin
Angewandte Chemie International Edition, 2011
Ubiquitin is a small signaling protein in cells and is highly conserved throughout the eukaryotes. Ubiquitin interacts with myriad partner proteins that contain one or more ubiquitin-binding domains (UBDs). To achieve multivalent binding with several UBDs, ubiquitins are often covalently linked by an isopeptide bond between the C-terminal carboxyl group of one ubiquitin and a primary amine in another; [1a,b] the two subunits in a di-ubiquitin are referred to as the proximal unit and the distal unit, respectively. All seven lysines and the N-terminus of ubiquitin can participate in the isopeptide bond. [2] In tandem, multiple ubiquitins can be linked up to form a poly(ubiquitin). Depending on the site of the linkage, di-and poly(ubiquitin)s can display distinct quaternary structures, which may account for their linkage-specific functions. [1a,b] Among the linkages, Lys11, Lys48, and Lys63-linked poly(ubiquitin)s are best characterized: Lys63-linked poly(ubiquitin) is involved in cellular events such as endocytosis and DNA repair, while both Lys11-and Lys48-linked poly(ubiquitin)s can signal for proteosomal degradation. [1a,b] In crystal, Lys48-linked diubiquitin mostly adopts a closed conformation, burying hydrophobic residues around Ile44 in both subunits; [3a,b, 4] Lys63-linked di-ubiquitin adopts an open extended structure; [5a,b, 6] while Lys11-linked di-ubiquitin displays intermediate subunit separation. [7, 8] Structural heterogeneity has also been observed for diand poly(ubiquitin)s with the same linkage. Lys48-linked diubiquitin has been crystallized in multiple forms, one of which actually adopts an open conformation (Figure S1a), [4] while in
Molecular Insights into Polyubiquitin Chain Assembly
Cell, 2001
and Biophysical Chemistry nal for proteolysis by 26S proteasomes (Chau et al., School of Medicine 1989; Finley et al., 1994), whereas Lys63-linked chains 2 Department of Biochemistry (K63-chains) are required for postreplicative DNA repair School of Public Health (Hofmann and Pickart, 1999; Spence et al., 1995), IKK 3 Howard Hughes Medical Institute activation (Deng et al., 2000), translational regulation Johns Hopkins University (Spence et al., 2000), and certain cases of ubiquitin-Baltimore, Maryland 21205 dependent endocytosis (Galan and Haguenauer-Tsapis, 1997). Blocking K63-chain assembly has no effect on proteasomal degradation in vivo (Spence et al., 1995), Summary and IKK activation can be reconstituted in vitro in the definitive absence of proteasomes (Deng et al., 2000). While the signaling properties of ubiquitin depend on IKK activation is selectively inhibited by free K63-chains, the topology of polyubiquitin chains, little is known and not K48-chains (Deng et al., 2000). Thus, while the concerning the molecular basis of specificity in chain precise signaling functions of K63-chains remain to be assembly and recognition. UEV/Ubc complexes have defined, it is highly unlikely that these chains signal been implicated in the assembly of Lys63-linked polyproteolysis by proteasomes. ubiquitin chains that act as a novel signal in postreplica-The ubiquitination of a protein substrate involves the tive DNA repair and IB␣ kinase activation. The crystal formation of an isopeptide bond between a substrate structure of the Mms2/Ubc13 heterodimer shows the lysine residue and the C-terminal carboxyl group of ubiactive site of Ubc13 at the intersection of two channels quitin Gly76. This reaction is accomplished through the that are potential binding sites for the two substrate sequential actions of three enzymes: a ubiquitin-activatubiquitins. Mutations that destabilize the heterodimer ing enzyme (E1), a ubiquitin-conjugating enzyme (E2 or interface confer a marked UV sensitivity, providing di-Ubc), and a ubiquitin-protein ligase (E3) (Hershko and rect evidence that the intact heterodimer is necessary Ciechanover, 1998). E1 forms an initial thioester bond for DNA repair. Selective mutations in the channels with the carboxyl terminus of ubiquitin in an ATP-depensuggest a molecular model for specificity in the asdent reaction. Ubiquitin is then transferred to the active sembly of Lys63-linked polyubiquitin signals. site cysteine of the E2. Isopeptide bond formation results from attack on the E2 bound ubiquitin by a lysine Introduction residue of the substrate. This final reaction usually requires the participation of an E3 that binds both the E2 Protein modification by ubiquitin serves a signaling ubiquitin thioester and the protein targeted for ubiquitifunction in diverse biological processes, including cell nation (Hershko and Ciechanover, 1998). Lysine resicycle progression (Koepp et al., 1999), oncogenesis dues within ubiquitin may also serve as substrates, lead-(Joazeiro and Weissman, 2000; Joazeiro et al., 1999), ing to the formation of diubiquitin and, eventually, and antigen presentation (Rock and Goldberg, 1999). polyubiquitin chains. While the overall folds of mono-The ability of ubiquitin to signal substrate proteolysis meric E2 enzymes, both alone and in complex with E3 by the 26S proteasome underlies many of ubiquitin's ligases, have been determined (Cook et al., 1992; Huang cellular functions (Hershko and Ciechanover, 1998). et al., 1999; Jiang and Basavappa, 1999; Worthylake However, ubiquitin also serves as a nonproteolytic siget al., 1998; Zheng et al., 2000), a plausible molecular nal in DNA repair (Jentsch et al., 1987; Spence et al., mechanism for catalysis or specificity in ubiquitin-sub-1995) and IB␣ kinase (IKK) activation (Chen et al., 1996). strate conjugation has not yet emerged. Furthermore, Ubiquitin conjugation also serves a signaling function the key question of how polyubiquitin chains are selecin endocytosis that leads to proteolysis in the lysosome/ tively linked through specific lysine residues has not vacuole (Hicke, 1999), and ubiquitin conjugation can previously been addressed. modulate the structural organization of multiprotein as-Ubiquitin E2 variant (UEV) proteins play a central role semblies (Kaiser et al., 2000). in the assembly of K63-linked polyubiquitin chains (Deng The biologically active ubiquitin signal frequently conet al., 2000; Hofmann and Pickart, 1999; Sancho et al., sists of a polyubiquitin chain in which successive ubiqui-1998; Ulrich and Jentsch, 2000). UEVs are similar in tins are joined through isopeptide bonds involving spesequence to E2s, but lack the active site cysteine resicific lysine residues of ubiquitin. In some cases, the fate due, indicating that UEVs must serve a fundamentally of a ubiquitin-conjugated protein can be correlated with different role in ubiquitin conjugation from that performed by canonical E2s (Broomfield et al., 1998; Sancho et al., 1998). The UEVs characterized to date func-4 Correspondence: cwolberg@jhmi.edu tion together with a canonical E2, Ubc13, in the 5 Present address: Laboratory of Synthetic Protein Chemistry, The Rockefeller University, New York, New York 10021. assembly of K63-chains (Deng et al., 2000; Hofmann receptors, transporters and channels. Trends Cell Biol. 9, 107-112. C. Ogata and R. Abramowitz of NSLS beamline X4A for technical Hofmann, R.M., and Pickart, C.M. (1999). Noncanonical MMS2support. This work was supported by the Howard Hughes Medical encoded ubiquitin-conjugating enzyme functions in assembly of Institute (C.W.), the NIH (GM60372, C.P.), and by a training grant novel polyubiquitin chains for DNA repair. Cell 96, 645-653. from NIEHS. Huang, L., Kinnucan, E., Wang, G., Beaudenon, S., Howley, P.M., Huibregtse, J.M., and Pavletich, N.P. (1999). Structure of an E6AP-