Insights into the Mechanism of Deubiquitination by JAMM Deubiquitinases from Cocrystal Structures of the Enzyme with the Substrate and Product

Abstract

AMSH, a conserved zinc metallo deubiquitinase, controls downregulation and degradation of cell-surface receptors mediated by the endosomal sorting complexes required for transport (ESCRT) machinery. It displays high specificity toward the Lys63-linked polyubiquitin chain, which is used as a signal for ESCRT-mediated endosomal–lysosomal sorting of receptors. Herein, we report the crystal structures of the catalytic domain of AMSH orthologue Sst2 from fission yeast, its ubiquitin (product)-bound form, and its Lys63-linked diubiquitin (substrate)-bound form at 1.45, 1.7, and 2.3 Å, respectively. The structures reveal that the P-side product fragment maintains nearly all the contacts with the enzyme as seen with the P portion (distal ubiquitin) of the Lys63-linked diubiquitin substrate, with additional coordination of the Gly76 carboxylate group of the product with the active-site Zn2+. One of the product-bound structures described herein is the result of an attempt to cocrystallize the diubiquitin substrate bound to an active site mutant presumed to render the enzyme inactive, instead yielding a cocrystal structure of the enzyme bound to the P-side ubiquitin fragment of the substrate (distal ubiquitin). This fragment was generated in situ from the residual activity of the mutant enzyme. In this structure, the catalytic water is seen placed between the active-site Zn2+ and the carboxylate group of Gly76 of ubiquitin, providing what appears to be a snapshot of the active site when the product is about to depart. Comparison of this structure with that of the substrate-bound form suggests the importance of dynamics of a flexible flap near the active site in catalysis. The crystal structure of the Thr319Ile mutant of the catalytic domain of Sst2 provides insight into structural basis of microcephaly capillary malformation syndrome. Isothermal titration calorimetry yields a dissociation constant (KD) of 10.2 ± 0.6 μM for the binding of ubiquitin to the enzyme, a value comparable to the KM of the enzyme catalyzing hydrolysis of the Lys63-linked diubiquitin substrate (∼20 μM). These results, together with the previously reported observation that the intracellular concentration of free ubiquitin (∼20 μM) exceeds that of Lys63-linked polyubiquitin chains, imply that the free, cytosolic form of the enzyme remains inhibited by being tightly bound to free ubiquitin. We propose that when AMSH associates with endosomes, inhibition would be relieved because of ubiquitin binding domains present on its endosomal binding partners that would shift the balance toward better recognition of polyubiquitin chains via the avidity effect.

Discovery of an Inhibitor of the Proteasome Subunit Rpn11

“…The Zn 2+ acts as a Lewis acid and increases the nucleophilic character of the bound water enough to allow hydrolytic cleavage of the isopeptide bond. 20, 24 …”

Ubiquitin Chains Modified by the Bacterial Ligase SdeA Are Protected from Deubiquitinase Hydrolysis

“…1–3 Several categories of human DUBs have been identified to date; these include the USP, UCH, OTU, MJD, and MINDY 4 families, cleaving ubiquitin linkages through a cysteine protease mechanism, and the JAMM family, utilizing a zinc metalloprotease mechanism. 5,6 Precise processing of ubiquitin chains by DUBs serves an important regulatory function in eukaryotes and is crucial for normal cellular function.…”

Structural Insight into Ubiquitin-Like Protein Recognition and Oligomeric States of JAMM/MPN+ Proteases

“…Most of these JAMMs require association in multisubunit complexes for full activity including: Rpn11 of the 26S proteasome (Pathare et al , 2014; Worden et al , 2014), CSN5 of the COP9 signalosome (Birol et al , 2014; Echalier et al , 2013; Lingaraju et al , 2014), and BRCC36 of the Abraxas (or BRCC36 isopeptidase) complex (Zeqiraj et al , 2015). HvJAMM1, a promiscuous DUB-like metalloprotease of the archaeon Haloferax volcanii (Hepowit et al , 2012), and AMSH/AMSH-LP, a eukaryotic DUB specific for Lys63-linked Ub chains (Sato et al , 2008; Shrestha et al , 2014), differ from most JAMMs by their ability to cleave Ubl/Ub-linkages independent of protein partners.…”

“…One of the limiting factors is that the only atomic level structure of a JAMM/MPN+ protease bound to its substrate is AMSH/AMSH-LP, a DUB specific for Lys63-linked Ub chains (Sato et al , 2008; Shrestha et al , 2014). Here we solved the X-ray crystal structures of an archaeal JAMM/MPN+ metalloprotease (PfJAMM1) alone and in complex with SAMP2.…”