Assembly and Specific Recognition of K29- and K33-Linked Polyubiquitin

Summary

Protein ubiquitination regulates many cellular processes via attachment of structurally and functionally distinct ubiquitin (Ub) chains. Several atypical chain types have remained poorly characterized because the enzymes mediating their assembly and receptors with specific binding properties have been elusive. We found that the human HECT E3 ligases UBE3C and AREL1 assemble K48/K29- and K11/K33-linked Ub chains, respectively, and can be used in combination with DUBs to generate K29- and K33-linked chains for biochemical and structural analyses. Solution studies indicate that both chains adopt open and dynamic conformations. We further show that the N-terminal Npl4-like zinc finger (NZF1) domain of the K29/K33-specific deubiquitinase TRABID specifically binds K29/K33-linked diUb, and a crystal structure of this complex explains TRABID specificity and suggests a model for chain binding by TRABID. Our work uncovers linkage-specific components in the Ub system for atypical K29- and K33-linked Ub chains, providing tools to further understand these unstudied posttranslational modifications.

Graphical Abstract

Highlights

• •

  The HECT E3 ligases UBE3C and AREL1 assemble K29- and K33-linked polyubiquitin, respectively

• •

  K29- and K33-linked chains adopt open conformations in solution

• •

  The N-terminal NZF1 domain of TRABID specifically recognizes K29/K33-diubiquitin

• •

  A structure of a K33 filament bound to NZF1 domains explains TRABID specificity

Abstract

Michel et al. reveal that UBE3C and AREL1 assemble K29- and K33-linked polyubiquitin, respectively, on substrates and as unanchored chains. They further identify a K29/K33-specific ubiquitin binding domain in TRABID and structurally characterize how TRABID recognizes K29/K33 filaments.