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FIGURE 2. Surface plasmon resonance of EDD UBA binding to ubiquitin and polyubiquitin chains. A, EDD UBA injected over monoubiquitin (Ub, open triangles), K48-linked diubiquitin (K48-Ub,, dashed line with filled rhom- bus), K48-linked tetraubiquitin (K48-Ub,, dashed line with filled squares), K63- linked diubiquitin (K63-Ub,, open rhombus), and K63-linked tetraubiquitin (K63-Ub,, open squares) surfaces. Experimental data points and the fitted binding curves are shown. The data were fit without cooperativity between sites (a Hill coefficient of 1) and normalized by the maximum response for each surface. Maximum responses were 770 (Ub), 1190 (K48-Ub,), 3340 (K48- Ub,), 2270 (K63-Ub,), and 3870 (K63-Ub,) in response units (RU). B, ubiquitin and polyubiquitin flowing over a UBA surface. Data and fitted curves are shown for monoubiquitin (Ub, open triangles), K48-linked diubiquitin (K48- Ub,, dashed line with filled rhombus), and N-to-C-linked diubiquitin (NC-Ub,, filled oval). The data were fit to allow cooperativity between sites with Hill coefficients of 0.99 (Ub), 0.87 (K48-Ub,), and 0.71 (NC-Ub,).

Figure 2 Surface plasmon resonance of EDD UBA binding to ubiquitin and polyubiquitin chains. A, EDD UBA injected over monoubiquitin (Ub, open triangles), K48-linked diubiquitin (K48-Ub,, dashed line with filled rhom- bus), K48-linked tetraubiquitin (K48-Ub,, dashed line with filled squares), K63- linked diubiquitin (K63-Ub,, open rhombus), and K63-linked tetraubiquitin (K63-Ub,, open squares) surfaces. Experimental data points and the fitted binding curves are shown. The data were fit without cooperativity between sites (a Hill coefficient of 1) and normalized by the maximum response for each surface. Maximum responses were 770 (Ub), 1190 (K48-Ub,), 3340 (K48- Ub,), 2270 (K63-Ub,), and 3870 (K63-Ub,) in response units (RU). B, ubiquitin and polyubiquitin flowing over a UBA surface. Data and fitted curves are shown for monoubiquitin (Ub, open triangles), K48-linked diubiquitin (K48- Ub,, dashed line with filled rhombus), and N-to-C-linked diubiquitin (NC-Ub,, filled oval). The data were fit to allow cooperativity between sites with Hill coefficients of 0.99 (Ub), 0.87 (K48-Ub,), and 0.71 (NC-Ub,).

Related Figures (7)

FIGURE 1. The UBA domain of EDD binds to ubiquitin and polyubiquitin. A, in vitro binding of the EDD and CblI-b UBA domains to ubiquitin agarose. B, in vitro binding of the EDD and CblI-b UBA domain to ubiquitin chains. C, calorimetric titration of the EDD UBA domain with ubiquitin. The panel shows the integrated heat released after correction for the heat of dilution (data points, squares) and the curve of best fit for binding toa single site.
FIGURE 1. The UBA domain of EDD binds to ubiquitin and polyubiquitin. A, in vitro binding of the EDD and CblI-b UBA domains to ubiquitin agarose. B, in vitro binding of the EDD and CblI-b UBA domain to ubiquitin chains. C, calorimetric titration of the EDD UBA domain with ubiquitin. The panel shows the integrated heat released after correction for the heat of dilution (data points, squares) and the curve of best fit for binding toa single site.
Data collection and refinement statistics
Data collection and refinement statistics
FIGURE 3. Structure of the EDD fragment containing the UBA domain and its comparison with other UBA/CUE domains. A, ribbon representation of the EDD UBA domain. The residues forming hydrophobic core are shown as sticks and labeled. Helices are labeled from a0 to a3. B, hydrophobic residues of the a1-a2 surface contact hydrophobic residues in the a0 helix. C, backbone overlay of the EDD UBA structure (yellow) with Dsk2 (green), Ede1 (dark blue), and Cue2 (light blue) structures. D, residues in the a1-a2 loop of EDD and Cue2. The loop conformation is strikingly similar in the UBA and CUE domain structures despite the different amino acids sequence. E, sequence alignment of the UBA domains from EDD, Dsk2, Ede1, and Ubx5 and CUE domain from Cue2. The a-helices and locations of amino acid substitutions in EDD that destabilize the protein (horizontal bars), that block ubiquitin binding (red X), or that have no effect (green circles) are shown.
FIGURE 3. Structure of the EDD fragment containing the UBA domain and its comparison with other UBA/CUE domains. A, ribbon representation of the EDD UBA domain. The residues forming hydrophobic core are shown as sticks and labeled. Helices are labeled from a0 to a3. B, hydrophobic residues of the a1-a2 surface contact hydrophobic residues in the a0 helix. C, backbone overlay of the EDD UBA structure (yellow) with Dsk2 (green), Ede1 (dark blue), and Cue2 (light blue) structures. D, residues in the a1-a2 loop of EDD and Cue2. The loop conformation is strikingly similar in the UBA and CUE domain structures despite the different amino acids sequence. E, sequence alignment of the UBA domains from EDD, Dsk2, Ede1, and Ubx5 and CUE domain from Cue2. The a-helices and locations of amino acid substitutions in EDD that destabilize the protein (horizontal bars), that block ubiquitin binding (red X), or that have no effect (green circles) are shown.
FIGURE 4. Crystal structure of the EDD UBA-ubiquitin complex. A, overall structure of the complex. Ubiquitin is in yellow, and the UBA domain is in purple B, UBA Val'®° binds the hydrophobic groove formed by lle**, His®, and Val’° of ubiquitin. C, UBA Leu** binds the groove formed by lle“, Arg*”, GIn*®, and Val”' of ubiquitin. D, Arg*? and Gln“? of ubiquitin form a network of intermolecular hydrogen bonds with Asn?" and Ser??° of EDD UBA. £, amide of Gly” of ubiquitir forms an intermolecular hydrogen bond with carbony] of Val?'® of EDD UBA via a bound water molecule. F, bound water molecule facilitates hydrogen bonding between amide of Leu’! of ubiquitin and side chain of Asn?'” of EDD UBA. The 2F,-F, electron density (1@ contour, omit map) for bound water molecules i shown in panels E and F.
FIGURE 4. Crystal structure of the EDD UBA-ubiquitin complex. A, overall structure of the complex. Ubiquitin is in yellow, and the UBA domain is in purple B, UBA Val'®° binds the hydrophobic groove formed by lle**, His®, and Val’° of ubiquitin. C, UBA Leu** binds the groove formed by lle“, Arg*”, GIn*®, and Val”' of ubiquitin. D, Arg*? and Gln“? of ubiquitin form a network of intermolecular hydrogen bonds with Asn?" and Ser??° of EDD UBA. £, amide of Gly” of ubiquitir forms an intermolecular hydrogen bond with carbony] of Val?'® of EDD UBA via a bound water molecule. F, bound water molecule facilitates hydrogen bonding between amide of Leu’! of ubiquitin and side chain of Asn?'” of EDD UBA. The 2F,-F, electron density (1@ contour, omit map) for bound water molecules i shown in panels E and F.
FIGURE 5. NMR mapping of EDD UBA-ubiquitin interactions in solution. A, 'H-'°N HSQC spectrum of the EDD UBA domain bound to ubiquitin. Signals are labeled with the one-letter amino acid code and residue number. Signals from asparagine and glutamine side chains are marked with horizontal lines. For clarity, peaks in the center of the spectrum are not labeled. B, plot of amide chemical shift changes of the UBA domain from EDD upon binding to monoubiquitin. The chemical shift changes were calculated as (AHN? + (0.2 < AN)?)'/2 The most affected residues are labeled. Secondary structure elements are shown above the plot. Plot (C), and mapping (D) of the chemical shift changes in EDD UBA upon ubiquitin binding. Gray indicates a large chemica' shift change; white indicates no change. and low sequence identity. The initial motivation for structural studies of EDD UBA was to explain ubiquitin binding by EDD, which lacks the conserved methionine residue found in the a1—a2 loop of other UBA domains. The structure revealed that the EDD possesses two extra residues in the a1—a2 loop, which positions a hydrophobic residue, valine, in place of the con- The structure also underlines the challenges of sequence alignment of UBA domains, which results from their small size
FIGURE 5. NMR mapping of EDD UBA-ubiquitin interactions in solution. A, 'H-'°N HSQC spectrum of the EDD UBA domain bound to ubiquitin. Signals are labeled with the one-letter amino acid code and residue number. Signals from asparagine and glutamine side chains are marked with horizontal lines. For clarity, peaks in the center of the spectrum are not labeled. B, plot of amide chemical shift changes of the UBA domain from EDD upon binding to monoubiquitin. The chemical shift changes were calculated as (AHN? + (0.2 < AN)?)'/2 The most affected residues are labeled. Secondary structure elements are shown above the plot. Plot (C), and mapping (D) of the chemical shift changes in EDD UBA upon ubiquitin binding. Gray indicates a large chemica' shift change; white indicates no change. and low sequence identity. The initial motivation for structural studies of EDD UBA was to explain ubiquitin binding by EDD, which lacks the conserved methionine residue found in the a1—a2 loop of other UBA domains. The structure revealed that the EDD possesses two extra residues in the a1—a2 loop, which positions a hydrophobic residue, valine, in place of the con- The structure also underlines the challenges of sequence alignment of UBA domains, which results from their small size
The EDD UBA-ubiquitin struc- ture explains how a smaller hydro- phobic residue, valine, can replace the larger methionine residue. Superposition of five complexes shows that the EDD UBA is slightly shifted relative to ubiquitin com- pared with other UBA/CUE domains (Fig. 7). In helix a1, this moves the side chain of Val'?° so that it occupies the same position as the methionine residue in other complexes with ubiquitin. The shift also leads to a larger distance between carbonyl of Val’’® and amide of Gly*’ of ubiquitin, which are unable to form the direct hydro- gen bond seen in other UBA-ubiq- uitin complexes. Interestingly, this intermolecular interaction is pre- served in the EDD UBA/ubiquitin FIGURE 6. Val'®° and Leu2”‘ of the UBA domain of EDD are required for ubiquitin binding. A, binding of mutant EDD UBA GST-fusion proteins to ubiquitir agarose. B, binding of K48-linked ubiquitin chains to immobilized GST-UBA mutants. C, binding of K63-linked ubiquitin chains to immobilized GST-UB/ mutants. D, mutated UBA residues in the x-ray structure of the UBA-ubiquitin complex. Residues required for binding are colored red (V196K, L224K), permissive residues are green (L214N, L218K), and residues that destabilized the UBA domain when mutated are colored gray (L189K, |190K, L206K).
The EDD UBA-ubiquitin struc- ture explains how a smaller hydro- phobic residue, valine, can replace the larger methionine residue. Superposition of five complexes shows that the EDD UBA is slightly shifted relative to ubiquitin com- pared with other UBA/CUE domains (Fig. 7). In helix a1, this moves the side chain of Val'?° so that it occupies the same position as the methionine residue in other complexes with ubiquitin. The shift also leads to a larger distance between carbonyl of Val’’® and amide of Gly*’ of ubiquitin, which are unable to form the direct hydro- gen bond seen in other UBA-ubiq- uitin complexes. Interestingly, this intermolecular interaction is pre- served in the EDD UBA/ubiquitin FIGURE 6. Val'®° and Leu2”‘ of the UBA domain of EDD are required for ubiquitin binding. A, binding of mutant EDD UBA GST-fusion proteins to ubiquitir agarose. B, binding of K48-linked ubiquitin chains to immobilized GST-UBA mutants. C, binding of K63-linked ubiquitin chains to immobilized GST-UB/ mutants. D, mutated UBA residues in the x-ray structure of the UBA-ubiquitin complex. Residues required for binding are colored red (V196K, L224K), permissive residues are green (L214N, L218K), and residues that destabilized the UBA domain when mutated are colored gray (L189K, |190K, L206K).
FIGURE 7. Overlay of five complexes of ubiquitin/UBL and UBA/CUE domains. The UBA domain of EDD binds to ubiquitin in a fashion similar tc other UBA and CUE domains. The structures are ubiquitin and UBA domain ot EDD (purple, this work), the ubiquitin and the UBA domain of Dsk2 (green, PDE accession code 1WR1), the ubiquitin, and the UBA domain of Ede1 (dark-blue PDB 2G3Q), ubiquitin and the CUE domain of Cue2 (teal, PDB 10TR), anda UBL domain complexed with the UBA domain of Dsk2 (coral, PDB 2BWE). The side chain of Val'?° of EDD UBA is shown.
FIGURE 7. Overlay of five complexes of ubiquitin/UBL and UBA/CUE domains. The UBA domain of EDD binds to ubiquitin in a fashion similar tc other UBA and CUE domains. The structures are ubiquitin and UBA domain ot EDD (purple, this work), the ubiquitin and the UBA domain of Dsk2 (green, PDE accession code 1WR1), the ubiquitin, and the UBA domain of Ede1 (dark-blue PDB 2G3Q), ubiquitin and the CUE domain of Cue2 (teal, PDB 10TR), anda UBL domain complexed with the UBA domain of Dsk2 (coral, PDB 2BWE). The side chain of Val'?° of EDD UBA is shown.
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ChemistryMedicineMacromolecular X-Ray CrystallographyProtein Secondary Structure PredictionIsothermal Titration CalorimetryMedical and Health Sciences
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