1H, 13C, and 15N resonance assignment of the ubiquitin-like domain from Dsk2p

Abstract

The ubiquitin-like domain (UBL) of yeast protein Dsk2p is widely believed to recognize and bind to ubiquitin receptors on the proteasome and, as part of Dsk2p, to bridge polyubiquitinated substrates and proteasomal degradation machinery. Here we report NMR resonance assignment for 1 H, 15 N, and 13 C nuclei in the backbone and side chains of the UBL domain of Dsk2p. This assignment will aid in NMR studies focused on understanding of Dsk2's interactions with proteasomal receptors and its role as a polyubiquitin shuttle in the ubiquitin-dependent proteasomal degradation as well as other cellular pathways. KeywordsDsk2p; ubiquitin-like domain; UBL; proteasome Biological contextDsk2p, a yeast protein comprising a N-terminal ubiquitin-like (UBL) domain, two stressinduced phosphoprotein 1 (STI1) domains, and a C-terminal ubiquitin-associated (UBA) domain, was first isolated from Saccharomyces cerevisiae as a suppressor of kar1 allele defective for spindle pole body duplication (Biggins et al. 1996;Funakoshi et al. 2002). Dsk2p belongs to a class of UBL-UBA proteins proposed to act as polyubiquitin shuttles in ubiquitinmediated protein degradation, the principal mechanism for the turnover of short-lived proteins in eukaryotes. Characteristic for the modular composition of these proteins, which include Rad23 and Dsk2 families, is the presence of a UBL domain at or near the N-terminus and a UBA domain at the C-terminus. The bi-functional nature of these proteins is based on the ability of the UBL domain to bind to the proteasome (Funakoshi et al. 2002) while the UBA domain can bind monomeric ubiquitin (monoUb) and polyubiquitin (polyUb) chains (Wilkinson et al. 2001).Dsk2p appears to play a similar role to Rad23, another UBL-UBA protein in yeast. Both proteins have been found to mediate the interaction between polyubiquitinated substrates and the proteasome (Funakoshi et al. 2002;Elsasser et al. 2004;Fujiwara et al. 2004;Verma et al. 2004;Ghaboosi and Deshaies 2007). However, Dsk2p is distinct from Rad23 that contains two UBA domains. In hHR23a, the human homologue of Rad23, the C-terminal UBA-2 has a rather low affinity for monoUb, but binds strongly and selectively to Lys48-linked polyUb chains (Varadan et al. 2004;Raasi et al. 2005;Varadan et al. 2005). Dsk2p's UBA, on the other hand, binds strongly already to monoUb and appears to bind polyUb chains nonselectively (Ohno et al. 2005;Raasi et al. 2005;Zhang et al. 2008). The UBL domains of both Dsk2p and Rad23 bind proteasomal subunit Rpn1, but only Dsk2p's UBL has been shown to interact with the Rpn10 subunit of the proteasome (Ishii et al. 2006). These structural and binding differences between Dsk2p and Rad23 suggest that these two proteins may differ in the specificity of their recognition by and association with the proteasome. A crystal structure of the UBL domain of Dsk2p (Fig. 1) has been solved by X-ray diffraction method (Lowe et al. 2006). However, very little is known with regard to Dsk2p's interactions with various proteasomal components and possi...

Together, Rpn10 and Dsk2 Can Serve as a Polyubiquitin Chain-Length Sensor

“…The chemical shifts, NOESY spectra, 15 N relaxation data, and residual dipolar couplings measured in Dsk2-UBL all are in general agreement (Fig. S4) with predictions from the crystal structure of Dsk2-UBL (PDB code 2BWF (Lowe et al, 2006)); therefore we conclude that the structure determined in the crystal form is largely preserved in solution, and our NMR signal assignments ((Chen et al, 2008) and Fig. S4) can be used to map the binding interactions.…”

“…Complete NMR resonance assignment for 1 H, 15 N, and 13 C nuclei of Dsk2-UBL domain was carried out previously (Chen et al, 2008). The chemical shifts, NOESY spectra, 15 N relaxation data, and residual dipolar couplings measured in Dsk2-UBL all are in general agreement (Fig.…”

Polyubiquitin-Photoactivatable Crosslinking Reagents for Mapping Ubiquitin Interactome Identify Rpn1 as a Proteasome Ubiquitin-Associating Subunit

“…At the end-point of titration, the concentrations are: [Rpn1] = 300 μM, [Dsk2] = 300 μM, [Ub2] = 4.8 mM. Shown is the spectral region containing CH3-Ile δ signals; the assignment of Dsk2 UBL signals is from (Chen et al, 2008). The red and green arrows highlight the signal shifts upon addition of Rpn1 391–642 and Ub 2 , respectively.…”