Strategies to trap enzyme-substrate complexes that mimic Michaelis intermediates during E3-mediated ubiquitin-like protein ligation Journal Article
| Authors: | Streich, F. C. Jr; Lima, C. D. |
| Article Title: | Strategies to trap enzyme-substrate complexes that mimic Michaelis intermediates during E3-mediated ubiquitin-like protein ligation |
| 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. © 2018, Springer Science+Business Media, LLC, part of Springer Nature. |
| Keywords: | ubiquitin; ligase; sumo; siz/pias; smt3; ubc9; structure; pcna; e3; ubiquitin-like protein; complex reconstitution; e2-conjugating enzyme |
| Journal Title: | Methods in Molecular Biology |
| Volume: | 1844 |
| ISSN: | 1064-3745 |
| Publisher: | Humana Press Inc |
| Date Published: | 2018-01-01 |
| Start Page: | 169 |
| End Page: | 196 |
| Language: | English |
| DOI: | 10.1007/978-1-4939-8706-1_12 |
| PROVIDER: | scopus |
| PUBMED: | 30242710 |
| PMCID: | PMC6390966 |
| DOI/URL: | |
| Notes: | Book Chapter 12 in "The Ubiquitin Proteasome System: Methods and Protocols" (ISBN: 978-1-4939-8705-4) -- Export Date: 1 November 2018 -- Source: Scopus |
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