Amino-terminal Dimerization, NRDP1-Rhodanese Interaction, and Inhibited Catalytic Domain Conformation of the Ubiquitin-specific Protease 8 (USP8)

Abstract

Ubiquitin-specific protease 8 (USP8) hydrolyzes mono and polyubiquitylated targets such as epidermal growth factor receptors and is involved in clathrin-mediated internalization. In 1182 residues, USP8 contains multiple domains, including coiled-coil, rhodanese, and catalytic domains. We report the first high-resolution crystal structures of these domains and discuss their implications for USP8 function. The amino-terminal domain is a homodimer with a novel fold. It is composed of two five-helix bundles, where the first helices are swapped, and carboxyl-terminal helices are extended in an antiparallel fashion. The structure of the rhodanese domain, determined in complex with the E3 ligase NRDP1, reveals the canonical rhodanese fold but with a distorted primordial active site. The USP8 recognition domain of NRDP1 has a novel protein fold that interacts with a conserved peptide loop of the rhodanese domain. A consensus sequence of this loop is found in other NRDP1 targets, suggesting a common mode of interaction. The structure of the carboxyl-terminal catalytic domain of USP8 exhibits the conserved tripartite architecture but shows unique traits. Notably, the active site, including the ubiquitin binding pocket, is in a closed conformation, incompatible with substrate binding. The presence of a zinc ribbon subdomain near the ubiquitin binding site further suggests a polyubiquitin-specific binding site and a mechanism for substrate induced conformational changes.The post-translational ubiquitylation system is composed of a cascade of E1, 2 E2, and E3 enzymes that activate and transfer ubiquitin or ubiquitin homologs to target proteins, including those targets that are membrane-anchored (1, 2). Modification by ubiquitin or ubiquitin-like molecules typically affects localization of the protein within the cell. For example, Lys-48-polyubiquitylated targets are recognized by the proteasome, which catalyzes complete target proteolysis (3). By affecting the abundance and/or half-life of signaling molecules, the ubiquitylation system can have dramatic effects on signal transduction pathways and play major roles in cellular biology. Degradation of integral membrane proteins can also be initiated by the ubiquitylation system but subsequent steps differ (4). Ubiquitylated receptors for example are rounded up by multivalent adaptors (e.g. AP2, epsin), which in turn recruit clathrin molecules that induce internalization through membrane budding. Dissociation of clathrin coats releases endosomes, which are either recycled back to the plasma membrane or fused with lysosomes, where membrane proteins undergo proteolytic degradation. Budding, uncoating, trafficking, and fusion events are influenced by the ubiquitylation state of the various intermediates, which is regulated not just by E3 ligases but by deubiquitylases as well.The human genome contains over 90 deubiquitylases, the specific biological roles of which are mostly unknown. Some deubiquitylases have been implicated in ubiquitin recycling, which typically can occur at the ...

Structure‐based mutagenesis of the substrate‐recognition domain of Nrdp1/FLRF identifies the binding site for the receptor tyrosine kinase ErbB3

“…1A). This structure superimposes with the recently reported structure of human Nrdp1C with a root mean square deviation of 0.62 Å for 123 Ca positions (Avvakumov et al 2006). An interesting feature of Nrdp1C is a negatively charged surface that is formed by the side chains of and encompasses the majority of one face of Nrdp1C (Fig.…”

Oligomerization of the Nrdp1 E3 Ubiquitin Ligase Is Necessary for Efficient Autoubiquitination but Not ErbB3 Ubiquitination

“…1). The carboxyl-terminal domain shows no homology to other proteins, exhibits a unique fold (34,35), and is responsible for ErbB3 binding. To explore the role of the coiled-coil domain in mediating ubiquitin ligase activity, we created several mutant forms of human Nrdp1 (Fig.…”

Mouse sperm acquire a new structure on the apical hook during epididymal maturation

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