doi: 10.1038/s41586-020-2000-y.
Epub 2020 Feb 12.
NEDD8 nucleates a multivalent cullin-RING-UBE2D ubiquitin ligation assembly
Affiliations
- PMID: 32051583
- PMCID: PMC7050210
- DOI: 10.1038/s41586-020-2000-y
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NEDD8 nucleates a multivalent cullin-RING-UBE2D ubiquitin ligation assembly
Nature.
2020 Feb.
Abstract
Eukaryotic cell biology depends on cullin-RING E3 ligase (CRL)-catalysed protein ubiquitylation1, which is tightly controlled by the modification of cullin with the ubiquitin-like protein NEDD82-6. However, how CRLs catalyse ubiquitylation, and the basis of NEDD8 activation, remain unknown. Here we report the cryo-electron microscopy structure of a chemically trapped complex that represents the ubiquitylation intermediate, in which the neddylated CRL1β-TRCP promotes the transfer of ubiquitin from the E2 ubiquitin-conjugating enzyme UBE2D to its recruited substrate, phosphorylated IκBα. NEDD8 acts as a nexus that binds disparate cullin elements and the RING-activated ubiquitin-linked UBE2D. Local structural remodelling of NEDD8 and large-scale movements of CRL domains converge to juxtapose the substrate and the ubiquitylation active site. These findings explain how a distinctive ubiquitin-like protein alters the functions of its targets, and show how numerous NEDD8-dependent interprotein interactions and conformational changes synergistically configure a catalytic CRL architecture that is both robust, to enable rapid ubiquitylation of the substrate, and fragile, to enable the subsequent functions of cullin-RING proteins.
Conflict of interest statement
Competing Interests Declaration
The authors declare no competing interests.
Figures
Gel images are representative of independent technical replicates (n=2); The symbols from graphs show the data from independent experiments (n = 2). a, Autoradiogram of SDS-PAGE gel showing products of ubiquitylation reactions under single encounter conditions for interaction of radiolabeled substrate (“medium β-catenin” substrate peptide derived from β-catenin) with neddylated CRL1β-TRCP, titrating UBE2D3 (referred to as UBE2D hereafter in legend). Each lane represented a single ubiquitylation reaction that was used to estimate the fraction of peptide that had been converted into ubiquitylated products as a function of UBE2D concentration. b, Plots of the fraction of substrate that had been converted to ubiquitylated products versus UBE2D concentration for ubiquitylation reactions containing either WT (as shown in a) or UBE2D S22R or H32A mutants. Various CRL1β-TRCP complexes were assayed that contained either WT neddylated CRL1β-TRCP (red), CRL1β-TRCP complexes modified by NEDD8 variants harboring I44A (orange), Q40E (green), “Ubiquitylizing” (L2Q K4F E14T D16E G63K G64E) substitutions (blue), CRL1β-TRCP modified by UB R72A that is competent for ligation to CUL1 (purple), or unmodified CRL1β-TRCP (CUL1 with the neddylation site K720R mutation, black). Duplicate data points from independent experiments performed with identical samples are shown and were fit to the Michaelis-Menten model to estimate the Km of UBE2D for CRL1β-TRCP using non-linear curve fitting (GraphPad Prism). c, Plots of the fraction of substrate that had been converted to ubiquitylated products versus UBE2D concentration for ubiquitylation reactions with various substrate peptides: derived from IκBα (but with a single acceptor Lys); derived from β-catenin; derived from β-catenin with different spacing between the phosphodegron motif and a potential acceptor Lys (a “medium β-catenin” substrate peptide with a 9-residue spacer between the β-catenin phosphodegron and acceptor matching the relative position of these moieties in IκBα, and “short β-catenin” substrate wherein the four residues between these moieties are too few to bridge the structurally-observed gap between the substrate receptor and UBE2D~UB active site); and a homogeneous UB linked-β-catenin generated by sortase-mediated transpeptidation wherein the only lysines are from UB. d, Autoradiogram of SDS-PAGE gel showing results from rapid quench-flow reactions under pre-steady state single encounter conditions for interaction of radiolabeled substrate (a “medium β-catenin” phosphopeptide) with CRL1β-TRCP. The representative raw data are from a reaction using WT UBE2D and WT neddylated CRL1β-TRCP and show time-resolved conjugation of increasing numbers of individual UB molecules. S0 = substrate with 0 UBs, S1 = substrate with 1 UB, S2 = substrate with 2 UBs, etc. e, Plots comparing various substrate peptides described in c, showing disappearance of unmodified substrate (S0) with black circles, and the appearance of mono-ubiquitylated substrate (S1) with gray triangles, in rapid quench-flow reactions all performed as in d and under single encounter conditions as in a. Duplicate data points from independent experiments performed with identical samples are shown. The data were fit to closed form equations (Mathematica) as previously described to obtain both the rates for the transfer of the first UB to substrate (kobsS0-S1) and of the second UB to the singly UB-modified substrate (kobsS1-S2) as well as their associated standard error (Table 1). f, Plots from experiments performed and analyzed as described in e, except with radiolabeled “medium β-catenin” peptide substrate, CRL1β-TRCP variants containing the indicated versions of CUL1-RBX1, and with either WT or indicated mutant versions of UBE2D.
a, Cryo EM density corresponding to substrate scaffolding regions of unneddylated CRL1β-TRCP is shown as surface with that encompassing RBX1’s RING and CUL1’s WHB domains, outlined for different 3D classes in different colors corresponding to percent of particles in that 3D class. b, same as in a, but with neddylated CRL1β-TRCP, with its surfaces outlined in different classes encompassing RBX1’s RING, CUL1’s WHB domain, and covalently modified NEDD8. c, Refined cryo EM density from CRL1β-TRCP reveals substrate scaffolding module bridging the substrate recruited to substrate receptor β-TRCP with the intermolecular cullin-RBX (C/R) domain, readily fitted with crystal structures of SKP1-β-TRCP [PDB: 1P22] and CUL1’s N-terminal domain and the C/R domain of CUL1-RBX1 [PDB: 1LDK]. d, Cartoon showing dynamics of NEDD8, its linked CUL1 WHB domain, and the RBX1 RING domain based on cryo EM data in b for substrate-bound neddylated CRL1β-TRCP, and model for varying locations of the RBX1 RING-bound UBE2D~UB relative to the substrate awaiting ubiquitylation. e, Left, cartoon representation of the catalytic architecture based on the cryo EM data shown in Figure 2, representing neddylated CRL1β-TRCP-catalyzed UB transfer from E2 UBE2D to an IκBα-derived substrate peptide. Right, semi-transparent version of the cartoon, highlighting the three modules (substrate scaffolding, catalytic, and activation modules), their constituents and locations establishing the catalytic architecture for substrate priming by neddylated CRL1β-TRCP and UBE2D.
Gel panels in this figure are representative from two independent experiments; n=2. a, Our strategy for trapping a mimic of the transient neddylated CRL E2~UB-substrate complex requires that the E2 UBE2D contain only a single cysteine at the active site. However, UBE2D contains three additional cysteines (Cys21, Cys107, Cys111). Standard Cys replacements by Ser or Ala severely compromised activity. Based on the structural locations of these cysteines, we presumed that their mutation hindered formation of the RING-activated, closed, active UBE2D~UB conformation–. We thus devised a systematic structure- and random-based approach to identify suitable replacements that qualitatively maintain wild-type levels of activity with neddylated CRLs. Structural analysis showed that Cys21 and Cys107 are in close proximity, such that mutation of both residues to Ala may generate a destabilizing cavity at this site. Combining UBE2D2 C107A with Cys21 mutated to Ile, Leu or Val to compensate for the reduced hydrophobic volume led to the identification of C21I C107A as a suitable version for testing of all other possible replacements for Cys111. A similar approach was taken for UBE2D3. A total of 48 different versions of UBE2D were tested to identify the C21I C107A C111D mutant for chemical trapping at the remaining active site Cys. b, Top, schematic of pulse-chase assay testing intrinsic activation of thioester-linked UBE2D~UB intermediates. Although this is often tested by monitoring RING-dependent discharge of UB from UBE2D to free lysine, RBX1 RING-dependent activity is limited in this assay due to sequence constraints imposed by the requirements for binding to partners other than UBE2D. Nonetheless, substrate-independent activation of UBE2D~UB can be readily visualized using CUL1 complexed with a previously-described hyperactive RBX1 N98R mutant, and high enzyme and lysine concentrations. UBE2D~UB generated in a pulse reaction was mixed with NEDD8-modified CUL1-RBX1 (shown here with N98R mutant) and free lysine, and UB discharge was monitored over time by Coomassie-stained SDS-PAGE as shown in representative gel on bottom demonstrating that standard Ser/Ala mutations of noncatalytic cysteines compromised activity (shown for C21A C107A C111S), while optimized version (C21I C107A C111D) retains wild-type like activity. c, Overview of the generation of our stable proxy for the phosphorylated IκBα substrate intermediate linked at a single atom, and comparison to the prior method employed to visualize non-canonical Lys sumoylation. d, Experiment validating our stable proxy for the UBE2D~UB-phosphorylated IκBα substrate intermediate linked at a single atom, based on the hypothesis that its simultaneous occupation of the binding sites for the UBE2D~UB intermediate and substrate should result in more potent inhibition of a neddylated CRL1β-TRCP-dependent substrate priming reaction compared to the individual constituents of the complex. e, Cryo EM reconstruction of neddylated CRL1β-TRCP2 (with full-length, dimeric β-TRCP2) bound to a mimic of the UBE2D2~UB-IκBα generated by adapting the method used previously to visualize non-canonical Lys sumoylation, where UB is isopeptide-bonded to a UBE2D L119K residue substitution, and a substrate Cys replacement for the acceptor is disulfide-bonded to the UBE2D2 catalytic Cys. This EM map visualizes the catalytic architecture of dimeric CRL1β-TRCP2 wherein the dimerization domain agrees well with the prior crystal structure, and its linked NEDD8 (encircled in yellow) is bound to the backside of UBE2D, but the donor UB (absent from region circled in orange) was not visible, presumably due to inadequacies of the method used to generate this mimic of the catalytic intermediate, in which the UB and substrate are not both simultaneously linked to the UBE2D catalytic Cys. Variations between the two protomers of the dimer also exacerbated sample heterogeneity. f, Cryo EM reconstruction of neddylated CRL1β-TRCP1ΔD (with monomeric version of β-TRCP1, from residue 175 to the C-terminus) bound to our newly devised proxy for the UBE2D3~UB-IκBα intermediate. The phoshpo-IκBα peptide-substrate-bound β-TRCP-SKP1-CUL1-RBX1-NEDD8-UBE2D portion of this map superimposes with the map for the dimeric complex shown in e, but here the entire complex is visible, including both the NEDD8 (encircled in yellow) and donor UB (encircled in orange). g, To further increase cryo EM sample homogeneity, we considered that the RBX1 RING sequence represents a compromise to meet requirements for its many different catalytic activities achieved with neddylation E2s, various UB carrying enzymes, and regulators including the inhibitor GLMN. Therefore, we mutationally introduced a second RBX1 linchpin residue (N98R) previously shown to improve neddylated CRL and UBE2D-dependent substrate priming at the expense of other RBX1-dependent functions (e.g. with UBE2M and UBE2R2). Shown is a Coommassie-stained SDS-PAGE gel from assay for intrinsic activity of UBE2D~UB, showing enhanced neddylated CRL-dependent activation of discharge to free lysine with the RBX1 N98R mutation. h,i, Cryo EM reconstructions of neddylated CRL1β-TRCP1ΔD with RBX1 N98R bound to our newly devised proxies for the UBE2D3~UB-IκBα and UBE2D2~UB-IκBα intermediates, the latter of which was pursued for high resolution electron microscopy (final reconstruction refined to 3.7Å resolution shown on right).
a, Cryo-EM image processing work chart. Ultimately, reconstruction of the data yielded a focused refinement at 3.46 Å resolution and a global refinement at 3.7 Å resolution that superimposes well with lower resolution maps obtained during attempts to visualize substrate priming with neddylated wild-type dimeric CRL1β-TRCP. b, 2D classes representing particles used for final reconstructions. c, Angular distribution of final reconstruction. d, Gold standard Fourier Shell Correlation curve showing overall resolution at 3.72Å at FSC=0.143. e, EM density map colored by local resolution. NEDD8, encircled in yellow, is the entity displaying the highest local resolution in the map.
a, Side-by-side comparison of relative RING domain locations in different CRL complexes after superposition of the C/R domains from the original CUL1-RBX1 structure (PDB ID 1LDJ, “Pre-Neddylation”, which data herein shows is dynamic, although the crystal structure likely captured the conformation allowing CAND1 binding and substrate receptor exchange), the structure representing the Neddylation reaction (PDB ID 4P5O), and a structure of a neddylated CUL5-RBX1 domain (PDB ID 3DQV, labeled “Post-Neddylation”, which revealed potential for neddylated CUL WHB and RBX1 RING domain conformational changes, and data herein shows is dynamic), and the structure presented here showing how the neddylated CUL1 WHB domain and RBX1 RING are harnessed in a catalytic architecture for “Active ubiquitylation”. RBX1’s Trp35 is highlighted to show it serving as a multifunctional platform for either the RING domain in different orientations, or for the E2-linked NEDD8 during neddylation. b, Superposition of the structures shown in a, highlighting different relative RING positions. c, Comparison of CUL WHB domain relative locations after superimposing their C/R domains (not shown). d, Cryo EM density from the neddylated CRL1β-TRCP-UBE2D~UB-substrate intermediate complex, showing patchiness of region corresponding to CUL1 “Helix-29”. This CUL1 region connecting the C/R and WHB domains is visible only as patchy density, whereas in prior cullin crystals this forms the rod-like Helix-29 continuing into the WHB domain. It seems CUL1’s Helix-29 dissolves into a flexible tether, which rationalizes the previously observed proteolytic sensitivity of this region in a neddylated CUL1-RBX1 complex, and enables the displacement and rotation required for placing the ensuing WHB domain and its linked NEDD8 at the center of the ubiquitylation complex.
a, Cryo EM density highlighting the relative placement of substrate degron and UBE2D~UB active site. The ~22Å distance between UBE2D~UB active site and the phosphodegron of β-TRCP-bound substrate requires at least 6 intervening residues in a substrate. b, Alignments for several reported β-TRCP substrates, highlighting the degron sequence (yellow) and nearby lysines (red). Also shown are sequences of peptide substrates with a single acceptor Lys that were used in kinetics analyses. The peptide sequences were derived from IκBα, and from β-catenin with varying spacers between phosphodegron and acceptor Lys: WT β-catenin peptide, “medium” β-catenin peptide with lysine corresponding to IκBα, and “short” β-catenin peptide with a lysine 5 residues upstream of the N-terminal phosphoSer in the degron, which would be too short to bridge the structurally-observed distance between the phosphodegron binding site on β-TRCP and UBE2D catalytic Cys in the ubiquitylation active site. c, Representative autoradiogram (n = 2) of SDS-PAGE gel showing products from indicated time points of ubiquitylation reactions under multi-turnover conditions with either neddylated or unneddylated CRL1β-TRCP and radiolabeled short β-catenin peptide substrate. The amount of short β-catenin peptide modified by neddylated CRL1β-TRCP and UBE2D is too low in the single-encounter ubiquitylation reaction to allow quantification of kinetic parameters, yet, product formation is apparent under multi-turnover conditions and shows that most products are heavily ubiquitylated. d, Plots fitting consumption of unmodified short β-catenin peptide substrate (S0) compared to formation of polyubiquitin chains with 5 or more UBs (S5+) from reactions as in panel c. The symbols show the data from independent experiments (n = 2 technical replicates).
a, NEDD8 and the catalytic module from the structure representing the neddylated CRL1β-TRCP-UBE2D~UB-IκBα intermediate, highlighting distinctive interactions between NEDD8 (yellow) and donor UB (orange) with UBE2D. b, Catalytic module from the neddylated CRL1β-TRCP-UBE2D~UB-IκBα intermediate, highlighting the covalently-linked proxy for the IκBα substrate’s acceptor in the active site relative to a superimposed representative prior crystal structure of an isolated RING-UBE2D~UB complex (grey, PDB: 4AP4),. In the inset, the density for the covalently-linked proxy for IκBα substrate’s acceptor is shown in red the active site. The chemical trap superimposes with consensus acceptors visualized in active sites of sumoylation and neddylation intermediates, where aromatic side-chains guide the lysine targets (blue and green, respectively),. However, UBE2D’s myriad substrates neither conform to a specific motif, nor do they or UBE2D display specific side-chains guiding lysine acceptors into the catalytic center. Instead, in the neddylated CRL1β-TRCP-UBE2D~UB-substrate complex, density from backbone atoms preceding the chemical proxy for the acceptor Lys corresponds to the aromatic guides in sumoylation and neddylation intermediates. c, Overview of assays for activation of intrinsic reactivity of UBE2D~UB intermediate. Top, schematic of pulse-chase assay for testing effects of UBE2D mutations on activation, monitoring UBE2D~UB discharge to free lysine activated by neddylated CUL-RBX1 compared to unneddylated or RING-like UBE4B controls. Bottom, sites of mutations shown as spheres on structure of UBE2D from cryo EM structure of neddylated CRL1β-TRCP-UBE2D~UB-substrate complex. Sphere colors reflect both the locations and the effects on UBE2D~UB discharge to free lysine. Sites of mutations with marginal or no effect are shown in cyan, whereas those with major effects are colored. Mutations causing major defects map to RBX1 RING-binding site (blue), the interaction surface with the donor UB (orange), and the interaction surface with NEDD8 (yellow). d, Representative Coommassie-stained SDS-PAGE gels (of two independent experiments) shown for reactions monitoring substrate-independent discharge of UBE2D~UB to free lysine, in presence of CUL1-RBX1 (N98R) that was either neddylated or unneddylated (K720R), with either WT or indicated UBE2D3 mutants at binding sites for backside-bound NEDD8 (S22R), the RBX1 RING (F62A), and the covalently-linked donor UB in the closed conformation (S108L). e, Same as in d except testing effect of NEDD8 Q40E, which would disrupt the activation module. f, Reactions performed as in d, except with indicated variants of UBE2D2, in reactions with CUL1-RBX1 (N98R) that was either neddylated or unneddylated (K720R), or with the optimized RING-like U-box domain from UBE4B as a reference. For mutations reporting on the catalytic conformation (G24K, T36K, M38K, A96K and D112K), representative gels are shown for two experiments. All other experiments were performed once. g, Comparison of β1/β2 -loop conformations after superimposing the indicated structures of NEDD8 and UB. The comparison suggests that while NEDD8 and UB can adopt both “loop in” and “loop out” conformations, donors linked to E2 active sites in RING activated complexes adopt the “loop in” conformation, and those bound to UBE2D backside adopt “loop out” conformations. h, Only a “loop out” conformation is compatible with the neddylated CRL activation module structure, because “loop in” conformation from the structures shown in g would prevent noncovalent interactions with CUL1 WHB domain (green). i, Only a “loop out” conformation is compatible for the CUL1-linked NEDD8 to bind the catalytic module, because “loop in” conformations in the structures shown in g would prevent noncovalent interactions with the UBE2D backside (cyan).
Gel panels in this figure are representative from two independent experiments; n=2 technical replicates. a, Schematic of a qualitative substrate priming assay for testing effects of mutations in neddylated CRL1β-TRCP or UBE2D on substrate priming, monitoring fluorescent UB transfer from UBE2D3 to the phosphorylated IκBα substrate. b, Scan of SDS-PAGE detecting fluorescent UB transferred to IκBα-derived substrate in qualitative assay for NEDD8 activation of substrate priming. c, as in b, showing effect on substrate priming of disrupting the activation module with the NEDD8 Q40E mutation. d, as in b, showing the effect on substrate priming of disrupting interactions between the activation and catalytic modules with NEDD8 I44A or UBE2D S22R mutation. e, as in b, showing the effect on substrate priming of disrupting interactions between the activation and substrate scaffolding modules, though CUL1 modification by a “Ubiquitylized” NEDD8 mutant with six residues swapped for UB counterparts (L2Q K4F E14T D16E G63K G64E). f, as in b, showing the effect of UBE2D H32A mutation at the interface between the catalytic and substrate scaffolding modules. g, Scheme of pulse-chase assay for testing effects of mutations in neddylated CRL1FBW7 or UBE2D on substrate priming. Assay monitors transfer of fluorescent UB from UBE2D to peptide substrate derived from phosphorylated Cyclin E (pCyE). h, Fluorescent scan detecting UB transferred to the pCyE substrate by neddylated CRL1FBW7 and the indicated mutants of UBE2D. i, Fluorescent scan detecting UB transferred to the pCyE substrate by UBE2D and indicated variants of neddylated (or ubiquitylated) CRL1FBW7. Experiment with unneddylated CRL1FBW7 use the K720R variant of CUL1 to prevent artifactual ubiquitylation. j, Scheme of pulse-chase assay for testing effects of mutations in neddylated CRL4CRBN or UBE2D on substrate priming, monitoring fluorescent UB transfer from UBE2D to the IKZF1/3 ZF2 substrate in the presence of the immunomodulatory drug pomalidomide. k, Fluorescent scan of assay validation, showing pomalidomide-dependence. l, Fluorescent scan detecting UB transferred to the IKZF substrate by CRL4CRBN, pomalidomide and the indicated variants of UBE2D. m-o, Fluorescent scan detecting UB transferred to the IKZF substrate by UBE2D and the indicated variants of neddylated (or ubiquitylated) CRL4CRBN with pomalidomide. Experiments with unneddylated CRL4CRBN use the K705R variant of CUL4A to prevent artifactual ubiquitylation.
a, Effect of CUL1 neddylation on CRL1β-TRCP-catalyzed UB transfer from E2 UBE2D to a radiolabeled β-catenin-derived peptide substrate. Plots show substrate remaining during pre-steady-state rapid quench-flow ubiquitylation reactions with saturating UBE2D3 and either unneddylated or neddylated CRL1β-TRCP. The symbols show the data from independent experiments (n = 2 technical replicates). b, Cartoon representing substrate priming by neddylated CRL1β-TRCP and UBE2D~UB. Inset shows transition state during ubiquitylation. c, Chemical mimic of the ubiquitylation intermediate, where surrogates for the active site of UBE2D, the C-terminus of UB, and the UB acceptor site on the IκBα-derived substrate peptide are simultaneously linked.
a, Cryo EM density representing neddylated CRL1β-TRCP-UBE2D~ubiquitin-IκBα substrate intermediate, wherein UBE2D~ubiquitin is activated and juxtaposed with substrate. b, The substrate scaffolding module connects β-TRCP-bound substrate to the intermolecular cullin-RBX (C/R) domain. c, The catalytic module consists of RBX1’s RING-UBE2D~UB in the canonical closed activated conformation, and additional density corresponding to the chemical surrogate for substrate undergoing ubiquitylation. d, NEDD8 and its covalently-linked CUL1 WHB domain form the activation module.
a, Cryo EM density highlighting noncovalent interfaces contributing to the catalytic architecture for neddylated CRL1β-TRCP-mediated UB transfer from UBE2D to a substrate. Circled regions correspond to interfaces within activation module, and between activation and catalytic, activation and substrate scaffolding and catalytic and substrate scaffolding modules shown in panels b-f. b, Close-up of intra-activation module interface, showing NEDD8’s buried polar residue Gln40 and Ile36/Leu71/Leu73 hydrophobic patch making noncovalent interactions with CUL1’s WHB domain adjacent to the isopeptide bond linking NEDD8 and CUL1. c, Close-up of interface between activation and catalytic modules showing key residues at interface between NEDD8 and UBE2D backside. d, Close-up highlighting NEDD8 residues in orange that differ in UB and are at interface with substrate scaffolding module. e, Close-up highlighting UBE2D His32 at interface with substrate scaffolding module. f, Close-up showing role of NEDD8 Loop-out conformation required for binding UBE2D.
a, Effects of indicated mutants within activation module, between activation and catalytic, activation and substrate scaffolding and substrate scaffolding and catalytic modules, alone or in combination, on catalytic efficiency of substrate priming as quantified by overall fold difference in kobs/Km versus wild-type neddylated CRL1β-TRCP and UBE2D-catalyzed ubiquitylation of a peptide substrate. Reactions with unneddylated CRL1β-TRCP serve as a reference, and used CUL1 K720R to prevent obscuring interpretation of results by artifactual UB transfer to CUL1 and resultant artifactual activation of substrate priming. Graphs show average value from two different experiments (technical replicates), for which curve fits and values are provided in Extended data Figure 1 and Extended Data Table 1. b, On their own, neddylated CRL1β-TRCP and UBE2D~UB are dynamic, and at an extreme their constituent proteins and/or domains may be substantially waving around. Mobile entities are harnessed in the neddylated CRL1β-TRCP-UBE2D~UB-substrate intermediate. There could be multiple routes to the catalytic architecture. It seems equally plausible that the UBE2D~UB intermediate would first encounter RBX1’s RING domain or NEDD8, either of which would raise the effective concentration for the other interaction. Likewise, noncovalent-binding between NEDD8 and its linked WHB domain, or with UBE2D’s backside, would stabilize NEDD8’s Loop-out conformation favoring the other interaction as well. Ultimately, NEDD8, the cullin, and the RBX1-bound UBE2D~UB intermediate make numerous interactions that synergistically establish a distinctive catalytic architecture placing UBE2D adjacent to β-TRCP.
Comment in
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Transfer of ubiquitin protein caught in the act.Nature. 2020 Feb;578(7795):372-373. doi: 10.1038/d41586-020-00325-9. Nature. 2020. PMID: 32066917 No abstract available.
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