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.

• MeSH
• interakční proteinové domény a motivy * MeSH
• katalytická doména MeSH
• konformace proteinů MeSH
• krystalografie rentgenová MeSH
• molekulární modely MeSH
• multigenová rodina MeSH
• poškození DNA * MeSH
• proteomika metody   MeSH
• Saccharomyces cerevisiae - proteiny chemie   genetika   metabolismus   MeSH
• Saccharomyces cerevisiae genetika   metabolismus   MeSH
• sekvence aminokyselin MeSH
• substrátová specifita MeSH
• ubikvitin metabolismus   MeSH
• vazba proteinů MeSH
• vazebná místa MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• DDI1 protein, S cerevisiae MeSH Prohlížeč
• Saccharomyces cerevisiae - proteiny MeSH
• ubikvitin MeSH

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.

• MeSH
• aminokyselinové motivy MeSH
• aspartátové proteasy chemie   metabolismus   MeSH
• HEK293 buňky MeSH
• konzervovaná sekvence MeSH
• krystalografie rentgenová MeSH
• lidé MeSH
• maloúhlový rozptyl MeSH
• mapování interakce mezi proteiny MeSH
• molekulární evoluce MeSH
• molekulární modely MeSH
• multimerizace proteinu MeSH
• polyubikvitin metabolismus   MeSH
• proteinové domény MeSH
• proteolýza MeSH
• roztoky MeSH
• Saccharomyces cerevisiae - proteiny chemie   metabolismus   MeSH
• Saccharomyces cerevisiae metabolismus   MeSH
• sekvence aminokyselin MeSH
• sekvenční analýza proteinů MeSH
• strukturní homologie proteinů * MeSH
• vazba proteinů MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• aspartátové proteasy MeSH
• DDI2 protein, human MeSH Prohlížeč
• polyubikvitin MeSH
• roztoky MeSH
• Saccharomyces cerevisiae - proteiny MeSH