Crystal Structure of a Josephin-Ubiquitin Complex

Abstract

The Josephin domain is a conserved cysteine protease domain found in four human deubiquitinating enzymes: ataxin-3, the ataxin-3-like protein (ATXN3L), Josephin-1, and Josephin-2. Josephin domains from these four proteins were purified and assayed for their ability to cleave ubiquitin substrates. Reaction rates differed markedly both among the different proteins and for different substrates with a given protein. The ATXN3L Josephin domain is a significantly more efficient enzyme than the ataxin-3 domain despite their sharing 85% sequence identity. To understand the structural basis of this difference, the 2.6 Å x-ray crystal structure of the ATXN3L Josephin domain in complex with ubiquitin was determined. Although ataxin-3 and ATXN3L adopt similar folds, they bind ubiquitin in different, overlapping sites. Mutations were made in ataxin-3 at selected positions, introducing the corresponding ATXN3L residue. Only three such mutations are sufficient to increase the catalytic activity of the ataxin-3 domain to levels comparable with that of ATXN3L, suggesting that ataxin-3 has been subject to evolutionary restraints that keep its deubiquitinating activity in check.

Ubiquitin Ser65 phosphorylation affects ubiquitin structure, chain assembly and hydrolysis

“…Ser65 in distal or proximal Ub is solvent-exposed in known structures of OTU DUBs, such as OTUB1 (Juang et al , 2012; Wiener et al , 2012) (Supplementary Fig S14B) and OTULIN (Keusekotten et al , 2013; Rivkin et al , 2013) (Supplementary Fig S14C). Similarly, Ser65 in the distal Ub is solvent-exposed in the Ataxin-3-like structure (Weeks et al , 2011) (Supplementary Fig S14D). In AMSH-LP, Ser65 of the proximal Ub is close to the enzyme (Sato et al , 2008) and may account for the observed reduced cleavage activity (Supplementary Fig S14E).…”

The Evolution and Functional Diversification of the Deubiquitinating Enzyme Superfamily

“…ATXN3L was duplicated from ATXN3 in the simian ancestor (figs. 1 B and 3 B ) and is the most effective ubiquitin cleaver of all Josephin domain-containing proteins, attributable to the optimization of two amino acid sites (Weeks et al. 2011).…”

SUMOylation of the brain-predominant Ataxin-3 isoform modulates its interaction with p97

“…(D) Far-UV CD spectra of unmodified Atx3 (blue), SUMOylated Atx3 (red), SUMO1 (green), an equimolar mixture of unmodified Atx3 and SUMO1 (uAtx3 + SUMO1 mix; grey) and the arithmetic sum of the independent spectra of unmodified Atx3 and SUMO 1 (uAtx3 + SUMO1; black). (E) Time course of cleavage of ubiquitin-6His substrate [31] by unmodified and SUMOylated Atx3. Triplicate assays were performed independently and the results of a representative experiment are presented.…”

“…Since aggregation is specific to certain brain regions, localization-30 dependent posttranslational modifications that differentially affect Atx3 might also contribute for MJD. 31 Methods: We combined in vitro and cellular approaches to address SUMOylation in the brain-predominant Atx3 32 isoform and assessed the impact of this posttranslational modification on Atx3 self-assembly and interaction 33 with its native partner, p97.…”