Diverse polyubiquitin interaction properties of ubiquitin-associated domains

Department of Biochemistry and Molecular Biology, Johns Hopkins School of Public Health, Baltimore, Maryland 21205, USA.
Nature Structural & Molecular Biology (Impact Factor: 11.63). 09/2005; 12(8):708-14. DOI: 10.1038/nsmb962
Source: PubMed

ABSTRACT The ubiquitin-associated (UBA) domain occurs frequently in proteins involved in ubiquitin-dependent signaling pathways. Although polyubiquitin chain binding is considered to be a defining feature of the UBA domain family, the generality of this property has not been established. Here we have surveyed the polyubiquitin interaction properties of 30 UBA domains, including 16 of 17 occurrences in budding yeast. The UBA domains sort into four classes that include linkage-selective polyubiquitin binders and domains that bind different chains (and monoubiquitin) in a nondiscriminatory manner; one notable class ( approximately 30%) did not bind any ubiquitin ligand surveyed. The properties of a given UBA domain are conserved from yeast to mammals. Their functional relevance is further suggested by the ability of an ectopic UBA domain to alter the specificity of a deubiquitylating enzyme in a predictable manner. Conversely, non-UBA sequences can modulate the interaction properties of a UBA domain.

1 Follower
  • Source
    • "To increase the binding affinity between F box proteins and their ubiquitinated substrates, we fused a ubiquitin-associated (UBA) domain from the soluble ubiquitin receptor Dsk2 or Rad23 to F box proteins (Figure 1A). These UBAs have a strong preference for polyubiquitin but bind both the K48-and K63- linked forms (Raasi et al., 2005; Sims et al., 2009). Using three tandem Flag epitopes as a linker, we fused the amino terminus of the F box protein Cdc4 to the tandem UBAs of Rad23 (Rad23-Fl-Cdc4). "
    [Show abstract] [Hide abstract]
    ABSTRACT: We have developed a technique, called Ubiquitin Ligase Substrate Trapping, for the isolation of ubiquitinated substrates in complex with their ubiquitin ligase (E3). By fusing a ubiquitin-associated (UBA) domain to an E3 ligase, we were able to selectively purify the polyubiquitinated forms of E3 substrates. Using ligase traps of eight different F box proteins (SCF specificity factors) coupled with mass spectrometry, we identified known, as well as previously unreported, substrates. Polyubiquitinated forms of candidate substrates associated with their cognate F box partner, but not other ligase traps. Interestingly, the four most abundant candidate substrates identified for the F box protein Saf1 were all vacuolar/lysosomal proteins. Analysis of one of these substrates, Prb1, showed that Saf1 selectively promotes ubiquitination of the unprocessed form of the zymogen. This suggests that Saf1 is part of a pathway that targets protein precursors for proteasomal degradation.
    Molecular cell 12/2013; 53(1). DOI:10.1016/j.molcel.2013.12.003 · 14.46 Impact Factor
  • Source
    • "The UBA2 domain of hHR23a binds preferentially to K48-linked chains (Raasi et al., 2005) and in a sandwich-like mode involving contacts with the hydrophobic patches of both Ub units and the linker region (Varadan et al., 2005). UBA2 also binds specifically but weakly to both monoUb (Mueller et al., 2004; Ryu et al., 2003) and K63-Ub 2 (Varadan et al., 2004). "
    [Show abstract] [Hide abstract]
    ABSTRACT: K11-linked polyubiquitin chains play important signaling and regulatory roles in both degradative and nonproteolytic pathways in eukaryotes. To understand the structural basis of how these chains are recognized and distinguished from other polyubiquitins, we determined solution structures of K11-linked diubiquitin (K11-Ub2) in the absence and presence of salt. These structures reveal that K11-Ub2 adopts conformations distinct from those of K48-linked or K63-linked chains. Importantly, our solution NMR and SANS data are inconsistent with published crystal structures of K11-Ub2. We found that increasing salt concentration compacts K11-Ub2 and strengthens interactions between the two Ub units. Binding studies indicate that K11-Ub2 interacts with ubiquitin-receptor proteins from both proteasomal and nonproteasomal pathways but with intermediate affinity and different binding modes than either K48-linked or K63-linked diubiquitin. Our data support the hypothesis that polyubiquitin chains of different linkages possess unique conformational and dynamical properties, allowing them to be recognized differently by downstream receptor proteins.
    Structure 07/2013; 21(7):1168-81. DOI:10.1016/j.str.2013.04.029 · 6.79 Impact Factor
  • Source
    • "The UBDs UBA (ubiquitin-associated) (Madura, 2002) and CUE (Ponting, 2000; Shih et al., 2003) interact with ubiquitin via a similar three a helix bundle, whereas other UBDs bind ubiquitin via a different tertiary structure (Randles and Walters, 2012; Searle et al., 2012). The affinity for both mono-or polyubiquitylated substrates and differently linked ubiquitin chains can vary greatly (Raasi et al., 2005; Dikic and Dö tsch, 2009; Husnjak and Dikic, 2012). "
    [Show abstract] [Hide abstract]
    ABSTRACT: Ubiquitin-binding domains (UBDs) differentially recognize ubiquitin (ub) modifications. Some of them specifically bind mono-ub, as has been shown for the CUE domain. Interestingly, so far no significant ubiquitin binding has been observed for the CUE domain of yeast Cue1p. Cue1p is receptor and activator of the ubiquitin-conjugating enzyme Ubc7p. It integrates Ubc7p into endoplasmic reticulum (ER) membrane-bound ubiquitin ligase complexes, and thus, it is crucial for ER-associated protein degradation (ERAD). Here we show that the CUE domain of Cue1p binds ubiquitin chains, which is pivotal for the efficient formation of K48-linked polyubiquitin chains in vitro. Mutations that abolish ubiquitin binding by Cue1p affect the turnover of ERAD substrates in vivo. Our data strongly imply that the CUE domain facilitates substrate ubiquitylation by stabilizing growing ubiquitin chains at the ERAD ubiquitin ligases. Hence, we demonstrate an unexpected function of a UBD in the regulation of ubiquitin chain synthesis.
Show more