The strand domain swap in the UbV
The β strand domain swap in the UbV.XR dimer appears to be a unique arrangement for a Ub-based dimer. In solution, free Ub has a KD of ∼5 mM for the monomer-dimer equilibrium, and the two subunits in the dimer adopt a range of relative orientations involving residues from Ub’s β sheet (Liu et al., 2012). Once UbV.XR dimer has formed, no shift to ZD 7288 sale is observed (Figure 4A), but the two dimeric subunits have some degree of flexibility relative to one another. Comparison to a range of Ub dimers involving isopeptide linkages with the N terminus or one of Ub’s Lys residues reveals that UbV.XR most closely resembles compact diUb chains linked through Lys6, Lys11, or Lys33 (RMSD ranging from 5.4 to 6.2 Å) (Bremm et al., 2010, Hospenthal et al., 2013, Matsumoto et al., 2010, Virdee et al., 2010), but modeling XR with any of these diUb chains superposed onto UbV.XR reveals potential clashes. We identified the G10A substitution in UbV.XR as being essential for UbV dimerization, but other substitutions may also contribute. Importantly, a phage-displayed dimeric UbV library can now be constructed using UbV.XR as a template, and such a library holds great potential for developing activators of other IAP proteins and other dimeric RING/U-box E3s.
Acknowledgments We thank W. Clark and A. Keith for in-house DNA sequencing, J. Gu for technical assistance in phage display, and Diamond Light Source for access to stations I03 and I04 (BAG allocation mx11651). W.Z. is supported by the Mitacs Elevate program. This work was supported by Cancer Research UK (C596/A23278) and European Research Council (ERC) under the European Union’s Horizon 2020 research and innovation programme (grant agreement n° 647849) awarded to D.T.H. and a Genome Canada Disruptive Innovation in Genomics grant (OGI-119) and a CQDM-OCE EXPLORE Project (#23927) awarded to S.S.S.
Introduction Eukaryotes use modifications with small protein modifiers to alter protein function or cellular fate [1, 2, 3]. The 8kDa protein ubiquitin is conjugated through its C-terminal glycine (Gly) to a lysine (Lys) residue of a target protein, thereby forming an isopeptide bond, through the consecutive activities of an E1 ubiquitin activating enzyme (UAE), E2 ubiquitin conjugating enzymes (UBC) and an E3 ligase (Figure 1) . E3 ligases confer specificity to the process through direct interactions with the substrate and by bringing the substrate close to the E2 (Figure 1) . A spectrum of ubiquitylation modifications has been described including mono-ubiquitylation, multi-ubiquitylation (multisite mono-ubiquitylation) as well as poly-ubiquitylation with different ubiquitin chain topologies . Depending on the nature of their ubiquitin modification, ubiquitylated proteins have different fates such as degradation by the 26S proteasome (Lys48-linked ubiquitin chains) or endocytosis from the plasma membrane to the vacuole (Lys63-linked chains), to name just two prominent examples . The type of ubiquitin modification and the substrate specificity of the ubiquitylation reaction depend on the respective E2 and E3 ubiquitin ligases, and both belong to protein families in plants and animals . Different E2 enzymes as well as E3 ligases can participate in a single ubiquitylation event and E3 ligases may be regulated by additional factors such as the recently characterized GLOMULIN proteins or their Arabidopsis thaliana (Arabidopsis) orthologue ABERRANT LATERAL ROOT FORMATION4 (ALF4) [,6,]. Analyses of plant genomes support the hierarchical nature of the ubiquitylation reaction with regard to its ability to gain increasing substrate specificity, for example, whereas the Arabidopsis thaliana genome encodes two E1 UAEs and 37 E2 UBCs, there are possibly over 1500 E3 ligases [4,8, 9, 10]. The high number of E3 ligases reveals the great importance of protein degradation during plant development as well as the importance of the general mechanisms regulating their assembly and functionality.