A unique E1-E2 interaction required for optimal conjugation of the ubiquitin-like protein NEDD8 - PubMed (original) (raw)

. 2004 Oct;11(10):927-35.

doi: 10.1038/nsmb826. Epub 2004 Sep 7.

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

Danny T Huang et al. Nat Struct Mol Biol. 2004 Oct.

Abstract

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.

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Figures

Figure 1

Figure 1

Ubc12’s N-terminal extension is important for function. a, Domain structure of Ubc12. Ubc12 has a unique 26-residue N-terminal extension conserved in Ubc12’s across species but not found in other E2s. Following this sequence is the ~150-residue E2 core conserved in all E2s. The position of Ubc12’s catalytic Cys is shown. b, NEDD8 conjugation involves the sequential action of NEDD8’s E1, the heterodimeric APPBP1-UBA3 complex, NEDD8’s E2, Ubc12, a RING E3 Rbx1, and cullin targets–,. c, NEDD8 conjugation to Cul1 was assayed by incubation with purified APPBP1-UBA3 (E1), Ubc12 or Ubc12ΔN lacking residues 2–26 (E2), Rbx1-Cul1 (E3-target complex, lanes 2, 5, 6) or Rbx1-Cul1 Lys720Arg (target complex mutated at NEDD8 modification site, lanes 7–8) as indicated. Lanes 1–4 are controls, lane 5 shows the complete reaction with wild-type Ubc12 and lane 6 shows the reaction with Ubc12ΔN. Lanes 7–8 show that neither Ubc12 nor Ubc12ΔN modify Cul1 with the Lys720Arg mutation. d, Immunoblot control for expression of Ubc12 and Ubc12ΔN in cell proliferation assay. e, Soft agar colony formation assay of NIH 3T3 cells expressing the mutant human CSF-1R[Tyr809Phe] infected with retroviruses empty (top left) or expressing wild type Ubc12 (top right) or the Ubc12ΔN mutant (bottom left) in the presence of human CSF-1. Relative % colony formation is based on controls expressing wild-type human CSF-1R as a reference for 100% (graph, bottom right).

Figure 2

Figure 2

Ubc12’s N-terminus is involved in E1 binding. a, Ubc12ΔN is impaired at forming a thioester complex with NEDD8. Ubc12~NEDD8 and Ubc12ΔN~NEDD8 thioester formation was examined as a function of E2 concentration. 30s time-points are shown for reactions involving 1 nM APPBP1-UBA3 and 25 nM, 100 nM, 500 nM, 1 μM, 5 μM and 10 μM Ubc12 or Ubc12ΔN, from left to right. b, Ubc12ΔN is competent for NEDD8 conjugation to Cul1. Top panel – Ubc12ΔN~NEDD8 thioester formation with increasing concentrations of the APPBP1-UBA3 E1 complex. Bottom panel – Ubc12ΔN-mediated NEDD8 conjugation to Cul-1, with increasing concentrations of APPBP1-UBA3, assayed as in the top panel but with the addition of Rbx1-Cul1. c, 1 mM Ubc12N26 peptide corresponding to the N-terminal 26 residues of Ubc12 inhibits APPBP1-UBA3-catalyzed Ubc12~NEDD8 thioester formation, but not thioester formation between Ubc12ΔN and NEDD8, or E1(ubiquitin)-catalyzed Ubc2p~ubiquitin or UbcH7~ubiquitin thioester formation, or E1(Sumo)-catalyzed Ubc9p~Sumo thioester formation. There is no effect of a peptide with identical composition but scrambled sequence (ScrambledN26). d, Dixon plot analyzing inhibition of APPBP1-UBA3-catalyzed Ubc12~NEDD8 thioester formation by the Ubc12N26 peptide. The Ubc12N26 peptide is a competitive inhibitor with a _K_i of 22±5 μM.

Figure 3

Figure 3

Electron density maps displayed over the Ubc12N26 peptide structure. a, Selenomethionine-scanning of Ubc12N26. Overlay of five different selenium anomalous difference Fourier maps contoured at 3.2σ, obtained from crystals containing Ubc12N26 peptides with each of the following residues substituted one-at-a-time with selenomethionine: Met1 (green map), Ile2 (blue map), Leu4 (magenta map), Leu7 (orange map), and Gln10 (red map). b, Fo-Fc map contoured at 3σ calculated after performing simulated annealing at 4000K on the model lacking the Ubc12N26 peptide.

Figure 4

Figure 4

Overall architecture of the APPBP1-UBA3-Ubc12N26 complex. Three views of the complex are shown in cartoon (top) and surface representations (bottom), each view with a 40–80° rotation around the y-axis as indicated. APPBP1 is shown in blue, UBA3 in red, and Ubc12N26 in cyan, and the position of the catalytic cysteine (C216A here) in green. The locations of the adenylation domain with its ATP binding site, the catalytic cysteine domain, the C-terminal domain (CTD), and the binding site for NEDD8’s globular domain are indicated. In the middle view, Cleft 1, which binds ATP is on the left, and Cleft 2, which binds the globular domain of NEDD8, is on the right. Residues 1 and 13 at the N- and C-termini of the visible portion of the Ubc12N26 peptide are labeled N and C, respectively. The “top” and “bottom” sides of UBA3’s Ubc12N26 binding groove are labeled.

Figure 5

Figure 5

The Ubc12N26 binding surface is conserved in UBA3s, but not in activating enzymes for other UBLs. The structure of Ubc12’s N-terminal peptide (cyan) is displayed in a surface representation of the Ubc12N26-binding domain of UBA3 (residues 9–205, 288–367), rotated ~90° in x relative to the middle orientation in Figure 4, for a direct view of the peptide contacts. a, The surface colored according to conservation among UBA3s from 8 species: H. sapiens, R. norvegicus, M. musculus, A. thaliana, C. elegans, D. melanogaster, D. rerio and S. pombe. b, The surface colored according to conservation among the corresponding region of activating enzymes for 8 different UBLs: human NEDD8, ubiquitin, Sumo and ISG15, S. cerevisiae Urm1p and Apg8p/Apg12p, and E. coli MoaD and ThiS. White equals 0% identity, yellow 25% identity, orange 50% identity, and brick 75–100% identity.

Figure 6

Figure 6

Contributions of individual residues from Ubc12’s N-terminal peptide to E1 binding. a, Stereoview of interactions between APPBP1-UBA3 and Ubc12’s N-terminal peptide, with the structure rotated ~90° in x relative to the middle orientation in Figure 4 for a direct view of the peptide contacts. APPBP1 is shown in blue, UBA3 in red with side-chains in yellow, and Ubc12N26 in cyan. Nitrogen atoms are highlighted in blue, oxygen atoms in red, sulfur atoms in green, and hydrogen bonds are dashed. b, Effects of alanine substitutions in Ubc12 on the relative binding affinity as a substrate for NEDD8’s E1, APPBP1-UBA3 plotted as (_K_m of wild-type Ubc12/_K_m of indicated variant of Ubc12). c, Effects of mutations in UBA3 on the relative binding affinity for Ubc12 as a substrate plotted as _K_m of Ubc12 (wild-type APPBP1-UBA3/APPBP1-indicated variant of UBA3).

Figure 7

Figure 7

Minimal length requirement for the linker between the E1 docking motif and the E2 core domain in Ubc12. a, Schematic diagram of insertion and deletion mutants used for these experiments. b, Sequences of Ubc12 linkers (residues 14–26 in wild-type Ubc12) for insertion and deletion mutants used for these experiments. Insertions and amino acid changes are highlighted in red, and deletions are denoted by dashes. c, _k_cat Ubc12 variant/_k_cat wild-type Ubc12 for the 1, 4, and 7 residue insertion mutants (In1, In4, In7), the 1, 4, 5, 6 and 7-residue deletion mutants (Δ1, Δ4, Δ5–1, Δ5–2, Δ6–1, Δ6–2, Δ7) and an additional alanine-containing mutant to control for sequence requirements (Δ4A3), as indicated. The inset shows Ubc12~NEDD8 thioester formation for wild-type Ubc12 and the 7-residue deletion mutant, Δ7, at concentrations of 0.05, 0.1, 0.2, 0.5, 1 and 5 μM.

Figure 8

Figure 8

Model for optimal positioning of Ubc12 in the E1 structure for formation of the Ubc12-NEDD8 thioester. APPBP1 is represented in blue, UBA3 in red, NEDD8 in yellow, Ubc12 in cyan, and catalytic cysteines in green. a, Ubc12’s interaction with APPBP1-UBA3 is bipartite: both Ubc12’s N-terminal peptide and conserved E2 core domain must bind the E1 simultaneously for optimal Ubc12~NEDD8 thioester formation. b, Deletions of 6 or more residues from the linker between Ubc12’s N-terminal docking peptide and E2 core domain are deleterious, either preventing docking of the N-terminal docking sequence (left panel) or the E2 core domain (right panel). The minimum length of 8 residues between Ubc12’s N-terminal 13-residue docking peptide and E2 core domain suggests that the E2 core domain binds Cleft 1 in the APPBP1-UBA3 structure.

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