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. [Display omitted] •Respective linkage-specific affimers recognize K6- or K33-/K11-linked chains•Structures of affimer:diUb complexes reveal mechanisms of linkage specificity•Improved affimers can be used in western blotting, confocal microscopy, and pull-downs•Pull-downs with K6-specific affimers reveal HUWE1 to be a major K6 ligase in cells Michel et al. describe linkage-specific tools for the study of atypical K6 and K33/K11 ubiquitin linkages. Affimers have applications in western blotting, confocal microscopy, and pull-downs. Using a K6-specific affimer, they reveal that HUWE1 is a major source of cellular K6 chains and decorates Mfn2 with K6 chains.