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.
Highlights
The ubiquitin system is primarily a signaling pathway whereby substrates tagged with various types of ubiquitin chains or ubiquitin-like (UBL) modifiers undergo different fates in the cell[1]
The focus of this study was to investigate the structure of yeast Ddi[1] and the interactions mediated by its UBL domain
Our isothermal titration calorimetry (ITC)-based affinity measurements deviate from the reported affinities measured by NMR (150 and 45 μM for the UBA and UBL, respectively34), Figure 5
Summary
The ubiquitin system is primarily a signaling pathway whereby substrates tagged with various types of ubiquitin chains or ubiquitin-like (UBL) modifiers undergo different fates in the cell[1]. In Saccharomyces cerevisiae, three ubiquitin receptors (Ddi[1], Rad[23], and Dsk2) have C-terminal UBA domains that bind ubiquitin and Lys48-linked polyubiquitin[3,4,5,6] These proteins bear an N-terminal UBL domain that binds Rpn[1] in the 19S proteasome subunit[7,8,9,10]. The linker includes a PEST motif and a phosphorylation site (T348) that regulates exocytosis[24] Consistent with these findings, Δddi[1] yeast cells show increased global protein secretion[23,25]. The active site aspartate is required for repression of protein secretion in yeast[25], and this phenotype can be inhibited by HIV protease inhibitors[28], strongly suggesting that this function of Ddi[1] is linked to its protease activity. In spite of all these studies, the overall function and substrate(s) of Ddi[1] protease domain remain elusive
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