Ubiquitin Linkage-Specific Affimers Reveal Insights into K6-Linked Ubiquitin Signaling.

Several ubiquitin chain types have remained unstudied, mainly because tools and techniques to detect these posttranslational modifications are scarce. Linkage-specific antibodies have shaped our understanding of the roles and dynamics of polyubiquitin signals but are available for only five out of eight linkage types. We here characterize K6- and K33-linkage-specific ...
"affimer" reagents as high-affinity ubiquitin interactors. Crystal structures of affimers bound to their cognate chain types reveal mechanisms of specificity and a K11 cross-reactivity in the K33 affimer. Structure-guided improvements yield superior affinity reagents suitable for western blotting, confocal fluorescence microscopy and pull-down applications. This allowed us to identify RNF144A and RNF144B as E3 ligases that assemble K6-, K11-, and K48-linked polyubiquitin in vitro. A protocol to enrich K6-ubiquitinated proteins from cells identifies HUWE1 as a main E3 ligase for this chain type, and we show that mitofusin-2 is modified with K6-linked polyubiquitin in a HUWE1-dependent manner.
Mesh Terms:
Amino Acid Motifs, Binding Sites, Carrier Proteins, Cell Line, Tumor, Cloning, Molecular, Crystallography, X-Ray, Escherichia coli, GTP Phosphohydrolases, Gene Expression, HEK293 Cells, HeLa Cells, Humans, Kinetics, Lysine, Mitochondrial Proteins, Models, Molecular, Molecular Probes, Protein Binding, Protein Interaction Domains and Motifs, Protein Processing, Post-Translational, Protein Structure, Secondary, Recombinant Proteins, Signal Transduction, Substrate Specificity, Ubiquitin, Ubiquitin-Protein Ligases, Ubiquitination
Mol. Cell
Date: Oct. 05, 2017
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