Abstract
Eukaryotic deubiquitinases are important regulators of ubiquitin signaling and can be subdivided into several structurally distinct classes. The ZUFSP family, with ZUP1 as its sole human member, has a modular architecture with a core catalytic domain highly active against the ubiquitin-derived peptide RLRGG, but not against ubiquitin itself. Ubiquitin recognition is conferred by additional non-catalytic domains, making full-length ZUP1 active against long K63-linked chains. However, non-mammalian ZUFSP family members contain different ubiquitin-binding domains in their N-terminal regions, despite their high conservation within the catalytic domain. Here, by working with representative ZUFSP family members from insects, fungi and plants, we show that different N-terminal domains are associated with different linkage preferences. Biochemical and structural studies suggest that the acquisition of two family-specific proximal domains have changed the default K48 preference of the ZUFSP family to the K63 preference observed in ZUP1 and its insect homolog. Additional N-terminal zinc finger domains promote chain cleavage without changing linkage-specificity.
Highlights
Eukaryotic deubiquitinases are important regulators of ubiquitin signaling and can be subdivided into several structurally distinct classes
Our results suggest that the K63 linkage specificity of metazoan ZUP1 members is caused by the interplay of the zUBD domain with the α2/ 3 helices, while the more distal ubiquitin-binding domains (UBDs) mostly contribute to avidity and the long-chain preference
As predicted by sequence analysis, all ubiquitin-binding domains of ZUP1 are absent from Mug[105], and no additional UBD candidates are apparent from the structure
Summary
Except for two short linker regions (149–153 and 189–193), the complete Mug[105] structure could be built from the electronic density by molecular replacement based on the catalytic domain of human ZUP1 (PDB:6EI1). The Mug[105] structure bears a striking resemblance to the ZUP1 catalytic core domain, with an RMSD of 1.7 Å over 215 residues (Fig. 1c). As predicted by sequence analysis, all ubiquitin-binding domains of ZUP1 are absent from Mug[105], and no additional UBD candidates are apparent from the structure. Asp-89 of Mug[105] corresponds to Asp-406 of ZUP1, which forms a salt bridge with Arg-72 of ubiquitin. Glu-109 of Mug[105] corresponds to Glu-428 of ZUP1, forming a salt bridge with Arg-74 of ubiquitin (Fig. 1d). Similar to what has been observed for ZUP1, these Mug[105] residues are required for. Unique reflections Multiplicity Completeness (%) Mean I/sigma(I) R-meas R-pim CC1/2 Reflections used in refinement Reflections used for R-free R-work/R-free (%) RMS (bonds) RMS (angles) Ramachandran favored (%) Ramachandran allowed (%) Ramachandran outliers (%) Rotamer outliers (%) Clashscore Average B-factor
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