Reconstitution and Structural Analysis of a HECT Ligase-Ubiquitin Complex via an Activity-Based Probe.
Ubiquitin activity-based probes have proven invaluable in elucidating structural mechanisms in the ubiquitin system by stabilizing transient macromolecular complexes of deubiquitinases, ubiquitin-activating enzymes, and the assemblies of ubiquitin-conjugating enzymes with ubiquitin ligases of the RING-Between-RING and RING-Cysteine-Relay families. Here, we demonstrate that an activity-based probe, ubiquitin-propargylamine, allows for the preparative ... reconstitution and structural analysis of the interactions between ubiquitin and certain HECT ligases. We present a crystal structure of the ubiquitin-linked HECT domain of HUWE1 that defines a catalytically critical conformation of the C-terminal tail of the ligase for the transfer of ubiquitin to an acceptor protein. Moreover, we observe that ubiquitin-propargylamine displays selectivity among HECT domains, thus corroborating the notion that activity-based probes may provide entry points for the development of specific, active site-directed inhibitors and reporters of HECT ligase activities.
Mesh Terms:
Amino Acid Sequence, Catalysis, Catalytic Domain, Cysteine, Humans, Models, Molecular, Pargyline, Propylamines, Protein Conformation, Structure-Activity Relationship, Substrate Specificity, Ubiquitin, Ubiquitin-Conjugating Enzymes, Ubiquitin-Protein Ligases, Ubiquitination
Amino Acid Sequence, Catalysis, Catalytic Domain, Cysteine, Humans, Models, Molecular, Pargyline, Propylamines, Protein Conformation, Structure-Activity Relationship, Substrate Specificity, Ubiquitin, Ubiquitin-Conjugating Enzymes, Ubiquitin-Protein Ligases, Ubiquitination
ACS Chem Biol
Date: Dec. 17, 2020
PubMed ID: 34403242
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