Ataxin-3, the disease protein in the neurodegenerative disorder Spinocerebellar 
Ataxia Type 3 or Machado Joseph disease, is a cysteine protease implicated in 
the ubiquitin proteasome pathway. It contains multiple ubiquitin binding sites 
through which it anchors polyubiquitin chains of different linkages that are 
then cleaved by the N-terminal catalytic (Josephin) domain. The properties of 
the ubiquitin interacting motifs (UIMs) in the C-terminus of ataxin-3 are well 
established. Very little is known, however, about how two recently identified 
ubiquitin-binding sites in the Josephin domain contribute to ubiquitin chain 
binding and cleavage. In the current study, we sought to define the specific 
contribution of the Josephin domain to the catalytic properties of ataxin-3 and 
assess how the topology and affinity of these binding sites modulate ataxin-3 
activity. Using NMR we modeled the structure of diUb/Josephin complexes and 
showed that linkage preferences are imposed by the topology of the two binding 
sites. Enzymatic studies further helped us to determine a precise hierarchy 
between the sites. We establish that the structure of Josephin dictates 
specificity for K48-linked chains. Site 1, which is close to the active site, is 
indispensable for cleavage. Our studies open the way to understand better the 
cellular function of ataxin-3 and its link to pathology.