Mechanism of Lys48-linked polyubiquitin chain recognition by the Mud1 UBA domain

Abstract

The ubiquitin-pathway associated (UBA) domain is a 40-residue polyubiquitin-binding motif. The Schizosaccharomyces pombe protein Mud1 is an ortholog of the Saccharomyces cerevisiae DNA-damage response protein Ddi1 and binds to K48-linked polyubiquitin through its UBA domain. We have solved the crystal structure of Mud1 UBA at 1.8 angstroms resolution, revealing a canonical three-helical UBA fold. We have probed the interactions of this domain using mutagenesis, surface plasmon resonance, NMR and analytical ultracentrifugation. We show that the ubiquitin-binding surface of Mud1 UBA extends beyond previously recognized motifs and can be functionally dissected into primary and secondary ubiquitin-binding sites. Mutation of Phe330 to alanine, a residue exposed between helices 2 and 3, significantly reduces the affinity of the Mud1 UBA domain for K48-linked polyubiquitin, despite leaving the primary binding surface functionally intact. Moreover, K48-linked diubiquitin binds a single Mud1 UBA domain even in the presence of excess UBA. We therefore propose a mechanism for the recognition of K48-linked polyubiquitin chains by Mud1 in which diubiquitin units are specifically recognized by a single UBA domain.

Figure 1

Crystal structure of the UBA domain from Mud1. (A) Cartoon representation of the asymmetric unit of the Mud1 UBA crystal structure formed by chain A (red) and B (blue). Labeled side chains are involved in domain–domain contacts or in Ub binding, and are drawn in stick mode. The sulfur anomalous difference map is shown as a blue contour at 10.0σ. (B) Electron density 2F_o–F_c map of Mud1 UBA crystal structure at a contour level of 1.0σ, showing a crystal contact between two ‘A' monomers in the crystal structure. Note the stacking of the side-chain phenyl groups of two Phe330 residues. Residues in a different symmetry-related molecule are tagged with an apostrophe.

Figure 2

Alignment of UBA and CUE domains found in multiple proteins. The SWISS-PROT ID code and residue numbers of the sequences are shown on the left. The secondary structure of Mud1 UBA is indicated by the boxes above the alignment. Residues with more than 70% conservation are shaded. Below, (^*) indicates conserved hydrophobic core residues and (#) indicates conserved hydrophobic residues exposed at the surface of the UBA domains. The SWISS-PROT ID codes for P62, SWA2 and P47 are Q13501, Q06677 and NSF1C, respectively.

Figure 3

Surface plasmon resonance of polyUb chains binding to immobilized biotin-Mud1 UBA. (A) Scaled experimental and fitted binding curves for the binding of polyUb chains to biotin-Mud1 UBA WT (25 RU). (B) Binding curves for the binding of polyUb chains to biotin-Mud1 UBA F330A (25 RU).

Figure 4

MonoUb-induced chemical shift perturbation studies of ¹⁵N-Mud1UBA WT and F330A. (A) Weighted-average chemical shift perturbations in backbone amide HSQC peaks of ¹⁵N-Mud1UBA WT (red) and F330A (blue) after addition of monoUb at saturation. For both molecules, the most affected regions are the conserved MGF loop (306–309) and the C-terminus of helix 3. The WT UBA shows additional shifts in helix 2. (B) Molecular surface representation of the chemical shift displacement of the Mud1 UBA backbone amide groups induced by monoUb on WT (center) and F330A (right). The intensity of the red color is proportional to the magnitude of the chemical shift displacement. A visual guide to the structure is shown as a cartoon rendered in ribbon and stick mode (left).

Figure 5

Mud1UBA-induced chemical shift perturbation studies of ¹⁵N-monoUb. (A) Weighted-average chemical shift perturbations in backbone amide HSQC peaks of ¹⁵N-monoUb after addition of Mud1 UBA at saturation. (B) Molecular surface representation of the chemical shift displacement of the Ub backbone and side-chain amide groups induced by Mud1 UBA on ¹⁵N-monoUb. The intensity of the red color is proportional to the magnitude of the chemical shift displacement. (C) The molecule in (B) was rotated by 120° to show shifts not associated with the hydrophobic patch.

Figure 6

Comparison of Ub₂ and monoUb-induced chemical shift perturbations of ¹⁵N-Mud1UBA WT and F330A. (A) Weighted average of the difference in chemical shifts induced in backbone amide HSQC peaks of ¹⁵N-Mud1UBA WT (red) and F330A (blue) obtained by subtracting ¹H and ¹⁵N chemical shift changes induced by monoUb from those induced by Ub₂. Resonances tagged with an asterisk (^*) could not be identified at the end of the titration. (B) Molecular surface representation of the weighted-average shift differences on Mud1 UBA WT (center) and F330A (right). The intensity of the red color is proportional to the magnitude of the chemical shift difference. A visual guide to the structure is shown as a cartoon rendered in ribbon and stick mode (left).

Figure 7

Molecular model for the interaction of Mud1 UBA with K48-linked Ub₂. (A) Intensity changes by cross-saturation of the ¹⁵N–¹H cross-peaks in 2H, ¹⁵N-Mud1 UBA in complex with unlabeled K48-Ub₂. (B) Primary (left) and secondary (right) binding sites on Mud1UBA as identified by NMR cross-saturation. Resonances showing intensity ratios <0.5 or 0.3 are displayed on the molecular surface of Mud1 UBA in light or dark red, respectively. (C) Closed conformation of Ub₂, based on the crystal structure obtained under basic conditions (PDB accession code 1AAR) (Cook et al, 1992). The hydrophobic patches on each Ub moiety interact with each other. The proximal and distal moieties of Ub₂ are colored in red and blue, respectively. (D) Open conformation of Ub₂, in equilibrium with closed conformation in solution. The two hydrophobic clusters formed by residues Leu8, Ile44, His68 and Val70 are available for binding of a single UBA domain via a primary (purple) and a secondary (blue) Ub-binding sites.