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 E2 Ubc9 -Ubl SUMO thioester mimetic that can be cross-linked to the Substrate PCNA at Lys164, a conjugation site that is only observed in the presence of E3 Siz1 . We describe two techniques to cross-link the E2 Ubc9 -Ubl SUMO 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 E2 Ubc9 -Ubl SUMO -PCNA complex with the E3 Siz1 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.