Human DNA-Damage-Inducible 2 Protein Is Structurally and Functionally Distinct from Its Yeast Ortholog

Abstract

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.

The Aspartic Protease Ddi1 Contributes to DNA-Protein Crosslink Repair in Yeast

“…On the other hand, the central part of Ddi1 encodes a highly conserved retroviral protease (RVP) domain with an HIV-like aspartic protease fold, emphasizing that Ddi1 is rather an unusual ubiquitin receptor Trempe et al, 2016). Consistent with the conservation of the RVP domain among eukaryotes, structural studies demonstrated that the Ddi1 dimer adopts a typical aspartic protease fold, strongly suggesting that the yeast and mammalian enzymes must be proteolytically active (Sirkis et al, 2006;Siva et al, 2016;Trempe et al, 2016). Although the Ddi1 proteolytic activity was previously addressed, it was not yet confirmed in vitro Trempe et al, 2016).…”

“…The RVP protease domain is the most conserved Ddi1 segment among eukaryotic orthologs, however its function remains the least understood. Structural analyses of the yeast and mammalian RVP domains strongly imply the presence of proteolytic activity, yet it has never been confirmed in vitro (Sirkis et al, 2006;Siva et al, 2016;Trempe et al, 2016). HDD is another evolutionally conserved domain with potential for DNA recognition based on its weak similarities to DNA binding domains .…”

Crystal structure of the retroviral protease‐like domain of a protozoal DNA damage‐inducible 1 protein

“…Structure comparison of LmDdi1-RVP with yDdi1-RVP and hDdi2-RVP The crystal structure information on the RVP domain of Ddi1 is available from S. cerevisiae (PDB Code: 2I1A, 4Z2Z) [27,28] and human (PDB Code: 4RGH) proteins [29]. The reported structures are similar to LmDdi1-RVP, but the flap information is either missing or limited.…”

“…It adopts an extended b-sheet conformation and forms an antiparallel hydrogen bonding network with one part of the flap [28]. In hDdi2-RVP also, only one flap that interacts with the symmetryrelated molecule has a well-defined electron density [29]. These observations suggest that the flap of the Ddi1-RVP domain has a general characteristic of being more mobile and disordered compared to that of HIV-1 PR and its density is observed only when its mobility is restricted due to crystal packing.…”

The yeast proteases Ddi1 and Wss1 are both involved in the DNA replication stress response

“…In this study, we tested the sensitivity of a double-deleted yeast strain lacking WSS1 and DDI1 to DNA-damaging chemicals and identified a hypersensitivity to hydroxyurea, a ribonucleotide reductase inhibitor that induces nucleotide depletion and arrests cells in the S phase. Based on previous studies and our structural characterization of yeast and mammalian Ddi1/2 Siva et al 2016), we performed rescue experiments with various deletions and single-site mutants of Ddi1 ( Fig. 1B) to assess the involvement of particular yeast Ddi1 domains in the replication stress response.…”