5OHL

K6-specific affimer bound to K6 diUb

Summary for 5OHL

• Entry DOI: 10.2210/pdb5ohl/pdb
• Descriptor: K6-specific affimer, Polyubiquitin-B, GLYCEROL, ... (5 entities in total)
• Functional Keywords: ubiquitin, cystatin, signaling protein
• Biological source: synthetic construct; More
• Cellular location: Ubiquitin: Cytoplasm : P0CG47
• Total number of polymer chains: 16
• Total formula weight: 180601.94

Authors

Michel, M.A.,Komander, D. (deposition date: 2017-07-17, release date: 2017-10-04, Last modification date: 2024-01-17)

Primary citation

Michel, M.A.,Swatek, K.N.,Hospenthal, M.K.,Komander, D.
Ubiquitin Linkage-Specific Affimers Reveal Insights into K6-Linked Ubiquitin Signaling.
Mol. Cell, 68:233-246.e5, 2017

PubMed Abstract: 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.

PubMed: 28943312
DOI: 10.1016/j.molcel.2017.08.020

Experimental method

X-RAY DIFFRACTION (2.5 Å)