Although Ddi1-like proteins are conserved among eukaryotes, their biological functions remain poorly characterized. Yeast Ddi1 has been implicated in cell cycle regulation, DNA-damage response, and exocytosis. By virtue of its ubiquitin-like (UBL) and ubiquitin-associated (UBA) domains, it has been proposed to serve as a proteasomal shuttle factor. All Ddi1-like family members also contain a highly conserved retroviral protease-like (RVP) domain with unknown substrate specificity. While the structure and biological function of yeast Ddi1 have been investigated, no such analysis is available for the human homologs. To address this, we solved the 3D structures of the human Ddi2 UBL and RVP domains and identified a new helical domain that extends on either side of the RVP dimer. While Ddi1-like proteins from all vertebrates lack a UBA domain, we identify a novel ubiquitin-interacting motif (UIM) located at the C-terminus of the protein. The UIM showed a weak yet specific affinity towards ubiquitin, as did the Ddi2 UBL domain. However, the full-length Ddi2 protein is unable to bind to di-ubiquitin chains. While proteomic analysis revealed no activity, implying that the protease requires other factors for activation, our structural characterization of all domains of human Ddi2 sets the stage for further characterization.

1st Faculty of Medicine Charles University Prague Katerinska 32 121 08 Prague 2 Czech Republic

Department of Biochemistry Faculty of Science Charles University Hlavova 8 128 00 Prague 2 Czech Republic

Department of Physical and Macromolecular Chemistry Faculty of Science Charles University Hlavova 8 128 00 Prague 2 Czech Republic

Gilead Sciences and IOCB Research Center Institute of Organic Chemistry and Biochemistry of the Academy of Sciences of the Czech Republic Flemingovo n 2 166 10 Prague 6 Czech Republic

Groupe de Recherche Axé sur la Structure des Protéines Department of Pharmacology and Therapeutics McGill University Montreal QC H3G 1Y6 Canada

Institute for Genetics University of Cologne Zülpicher Str 47a 50647 Cologne Germany

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Structural studies of the yeast DNA damage-inducible protein Ddi1 reveal domain architecture of this eukaryotic protein family