The eukaryotic Ddi1 family is defined by a conserved retroviral aspartyl protease-like (RVP) domain found in association with a ubiquitin-like (UBL) domain. Ddi1 from Saccharomyces cerevisiae additionally contains a ubiquitin-associated (UBA) domain. The substrate specificity and role of the protease domain in the biological functions of the Ddi family remain unclear. Yeast Ddi1 has been implicated in the regulation of cell cycle progression, DNA-damage repair, and exocytosis. Here, we investigated the multi-domain structure of yeast Ddi1 using X-ray crystallography, nuclear magnetic resonance, and small-angle X-ray scattering. The crystal structure of the RVP domain sheds light on a putative substrate recognition site involving a conserved loop. Isothermal titration calorimetry confirms that both UBL and UBA domains bind ubiquitin, and that Ddi1 binds K48-linked diubiquitin with enhanced affinity. The solution NMR structure of a helical domain that precedes the protease displays tertiary structure similarity to DNA-binding domains from transcription regulators. Our structural studies suggest that the helical domain could serve as a landing platform for substrates in conjunction with attached ubiquitin chains binding to the UBL and UBA domains.

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

Department of Biochemistry Faculty of Science Charles University Hlavova 8 120 00 Prague 2 Czech Republic

Department of Physical and Macromolecular Chemistry Faculty of Science Charles University Hlavova 8 120 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 Biochemistry McGill University 3649 Promenade Sir William Osler Montreal QC H3G 0B1 Canada

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