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Strategies to Trap Enzyme-Substrate Complexes that Mimic Michaelis Intermediates During E3-Mediated Ubiquitin-Like Protein Ligation

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The Ubiquitin Proteasome System

Part of the book series: Methods in Molecular Biology ((MIMB,volume 1844))

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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Acknowledgments

This work was supported in part by GM065872 and GM118080 (NIH/NIGMS, C.D.L) and P30CA008748 (NIH/National Cancer Institute). The content is the authors’ responsibility and does not represent the official views of the NIH. C.D.L is a Howard Hughes Medical Institute Investigator.

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Authors and Affiliations

  1. Structural Biology Program, Sloan Kettering Institute, Memorial Sloan Kettering Cancer Center, 1275 York Ave, New York, NY, USA

    Frederick C. Streich Jr. & Christopher D. Lima

  2. Howard Hughes Medical Institute, 1275 York Ave, New York, NY, USA

    Christopher D. Lima

Authors
  1. Frederick C. Streich Jr.
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  2. Christopher D. Lima
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Correspondence to Christopher D. Lima .

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Editors and Affiliations

  1. Department of Biochemistry and Molecular Biology, Michael Smith Laboratories, University of British Columbia, Vancouver, BC, Canada

    Thibault Mayor

  2. Department of Chemistry and Biochemistry, University of Nevada Las Vegas, Las Vegas, NV, USA

    Gary Kleiger

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Streich, F.C., Lima, C.D. (2018). Strategies to Trap Enzyme-Substrate Complexes that Mimic Michaelis Intermediates During E3-Mediated Ubiquitin-Like Protein Ligation. In: Mayor, T., Kleiger, G. (eds) The Ubiquitin Proteasome System. Methods in Molecular Biology, vol 1844. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-8706-1_12

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  • DOI: https://doi.org/10.1007/978-1-4939-8706-1_12

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