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 Å 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.