Mechanism of Lys48-linked polyubiquitin chain recognition by the Mud1 UBA domain.
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.
Trempe
Jean-François
JF
Laboratory of Molecular Biophysics, University of Oxford, Oxford, UK.
Brown
Nicholas R
NR
Lowe
Edward D
ED
Gordon
Colin
C
Campbell
Iain D
ID
Noble
Martin E M
ME
Endicott
Jane A
JA
D000595:Amino Acid Sequence; D002352:Carrier Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D008239:Lysine / Q000235:genetics / Q000378:metabolism*; D008958:Models, Molecular; D008969:Molecular Sequence Data; D019906:Nuclear Magnetic Resonance, Biomolecular; D025821:Polyubiquitin / Q000737:chemistry / Q000378:metabolism*; D011485:Protein Binding; D020836:Protein Structure, Quaternary; D017434:Protein Structure, Tertiary; D012568:Schizosaccharomyces / Q000235:genetics / Q000378:metabolism*; D029702:Schizosaccharomyces pombe Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D016415:Sequence Alignment; D020349:Surface Plasmon Resonance
D002352:Carrier Proteins; C504973:Mud1 protein, S pombe; D029702:Schizosaccharomyces pombe Proteins; D025821:Polyubiquitin; D008239:Lysine