title: Structural studies of the yeast DNA damage-inducible protein Ddi1 reveal domain architecture of this eukaryotic protein family.
abstract: 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.
authors.affiliation: Groupe de Recherche Axé sur la Structure des Protéines, Department of Biochemistry, McGill University, 3649 Promenade Sir William Osler, Montreal, QC, H3G 0B1, Canada.
authors.forename: Jean-François
authors.initials: JF
authors.lastname: Trempe
authors.affiliation: 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.|Department of Biochemistry, Faculty of Science, Charles University, Hlavova 8, 120 00 Prague 2, Czech Republic.
authors.forename: Klára Grantz
authors.initials: KG
authors.lastname: Šašková
authors.affiliation: 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.|Department of Biochemistry, Faculty of Science, Charles University, Hlavova 8, 120 00 Prague 2, Czech Republic.|First Faculty of Medicine, Charles University in Prague, Katerinska 32, 121 08, Prague 2, Czech Republic.
authors.forename: Monika
authors.initials: M
authors.lastname: Sivá
authors.affiliation: Groupe de Recherche Axé sur la Structure des Protéines, Department of Biochemistry, McGill University, 3649 Promenade Sir William Osler, Montreal, QC, H3G 0B1, Canada.
authors.forename: Colin D H
authors.initials: CD
authors.lastname: Ratcliffe
authors.affiliation: 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.
authors.forename: Václav
authors.initials: V
authors.lastname: Veverka
authors.affiliation: Groupe de Recherche Axé sur la Structure des Protéines, Department of Biochemistry, McGill University, 3649 Promenade Sir William Osler, Montreal, QC, H3G 0B1, Canada.
authors.forename: Annabelle
authors.initials: A
authors.lastname: Hoegl
authors.affiliation: Groupe de Recherche Axé sur la Structure des Protéines, Department of Biochemistry, McGill University, 3649 Promenade Sir William Osler, Montreal, QC, H3G 0B1, Canada.
authors.forename: Marie
authors.initials: M
authors.lastname: Ménade
authors.affiliation: Groupe de Recherche Axé sur la Structure des Protéines, Department of Biochemistry, McGill University, 3649 Promenade Sir William Osler, Montreal, QC, H3G 0B1, Canada.
authors.forename: Xin
authors.initials: X
authors.lastname: Feng
authors.affiliation: Groupe de Recherche Axé sur la Structure des Protéines, Department of Biochemistry, McGill University, 3649 Promenade Sir William Osler, Montreal, QC, H3G 0B1, Canada.
authors.forename: Solomon
authors.initials: S
authors.lastname: Shenker
authors.affiliation: 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.|Department of Physical and Macromolecular Chemistry, Faculty of Science, Charles University, Hlavova 8, 120 00 Prague 2, Czech Republic.
authors.forename: Michal
authors.initials: M
authors.lastname: Svoboda
authors.affiliation: 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.
authors.forename: Milan
authors.initials: M
authors.lastname: Kožíšek
authors.affiliation: 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.|Department of Biochemistry, Faculty of Science, Charles University, Hlavova 8, 120 00 Prague 2, Czech Republic.
authors.forename: Jan
authors.initials: J
authors.lastname: Konvalinka
authors.affiliation: Groupe de Recherche Axé sur la Structure des Protéines, Department of Biochemistry, McGill University, 3649 Promenade Sir William Osler, Montreal, QC, H3G 0B1, Canada.
authors.forename: Kalle
authors.initials: K
authors.lastname: Gehring
mesh_terms: D000595:Amino Acid Sequence; D001665:Binding Sites; D020134:Catalytic Domain; D018360:Crystallography, X-Ray; D004249:DNA Damage; D008958:Models, Molecular; D005810:Multigene Family; D011485:Protein Binding; D011487:Protein Conformation; D054730:Protein Interaction Domains and Motifs; D040901:Proteomics / Q000379:methods; D012441:Saccharomyces cerevisiae / Q000235:genetics* / Q000378:metabolism*; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D013379:Substrate Specificity; D025801:Ubiquitin / Q000378:metabolism
chemical_list: C108125:DDI1 protein, S cerevisiae; D029701:Saccharomyces cerevisiae Proteins; D025801:Ubiquitin