understanding the role of the josephin domain in the polyub binding and cleavage properties of ataxin-3理解josephin的作用域在polyub绑定和乳沟ataxin-3的属性.pdfVIP

Understanding the Role of the Josephin Domain in the PolyUb Binding and Cleavage Properties of Ataxin-3 1. 2. 3 3 2 Giuseppe Nicastro , Sokol V. Todi , Ezgi Karaca , Alexandre M. J. J. Bonvin , Henry L. Paulson , Annalisa Pastore1* 1 National Institute for Medical Research, Medical Research Council, London, United Kingdom, 2 Department of Neurology, University of Michigan, Ann Arbor, Michigan, United States of America, 3 Science Faculty, Bijvoet Center for Biomolecular Research, Utrecht University, Utrecht, The Netherlands Abstract 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. Si