Chapter title |
Strategies to Trap Enzyme-Substrate Complexes that Mimic Michaelis Intermediates During E3-Mediated Ubiquitin-Like Protein Ligation
|
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Chapter number | 12 |
Book title |
The Ubiquitin Proteasome System
|
Published in |
Methods in molecular biology, September 2018
|
DOI | 10.1007/978-1-4939-8706-1_12 |
Pubmed ID | |
Book ISBNs |
978-1-4939-8705-4, 978-1-4939-8706-1
|
Authors |
Frederick C. Streich, Christopher D. Lima, Streich, Frederick C., Lima, Christopher D. |
Abstract |
Most cellular functions rely on pathways that catalyze posttranslational modification of cellular proteins by ubiquitin (Ub) and ubiquitin-like (Ubl) proteins. Like other posttranslational modifications that require distinct writers, readers, and erasers during signaling, Ub/Ubl pathways employ distinct enzymes that catalyze Ub/Ubl attachment, Ub/Ubl recognition, and Ub/Ubl removal. Ubl protein conjugation typically relies on parallel but distinct enzymatic cascades catalyzed by an E1-activating enzyme, an E2 carrier protein, and an E3 ubiquitin-like protein ligase. One major class of E3, with ca. 600 members, harbors RING or the RING-like SP-RING or Ubox domains. These RING/RING-like domains bind and activate the E2-Ubl thioester by stabilizing a conformation that is optimal for nucleophilic attack by the side chain residue (typically lysine) on the substrate. These RING/RING-like domains typically function together with other domains or protein complexes that often serve to recruit particular substrates. How these RING/RING-like E3 domains function to activate the E2-Ubl thioester while engaged with substrate remains poorly understood. We describe a strategy to generate and purify a unique E2Ubc9-UblSUMO thioester mimetic that can be cross-linked to the SubstratePCNA at Lys164, a conjugation site that is only observed in the presence of E3Siz1. We describe two techniques to cross-link the E2Ubc9-UblSUMO thioester mimetic active site to the site of modification on PCNA and the subsequent purification of these complexes. Finally, we describe the reconstitution and purification of the E2Ubc9-UblSUMO-PCNA complex with the E3Siz1 and purification that enabled its crystallization and structure determination. We think this technique can be extended to other E2-Ubl-substrate/E3 complexes to better probe the function and specificity of RING-based E3 Ubl ligases. |
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