Design and Semisynthesis of Ubiquitin Extension Probes for Structural Analysis of Cullin1-Mediated Substrate Polyubiquitination

Abstract

Chemical trapping strategies have recently emerged as powerful approaches for investigating the structural dynamics of E3 ligase-catalyzed substrate ubiquitination. However, current ubiquitination-derived probes are limited to studying substrate mono- or diubiquitination events. Probes capable of investigating how E3 ligases accommodate E2–Ub conjugates and ubiquitinated substrates to generate longer ubiquitin chains remain unexplored. In this work, we report the development of two Cullin1 E3 ligase (CRL1)-dependent probes, Extension Probe^Ub2 and Extension Probe^Ub4, which mimic transient intermediates formed during CRL1-catalyzed K48-linked diubiquitin and tetraubiquitin chain formation on substrate p27. Notably, a chemoenzymatic semisynthetic strategy was devised to generate Extension Probe^Ub4, involving the enzymatic conjugation of a preformed K48-linked diubiquitin to a synthetic Ub-p27-degron construct using the E2 conjugating enzyme UBE2K. Both Extension Probe^Ub2 and Extension Probe^Ub4 formed stable complexes with N8-CRL1^{Skp1/Skp2/Cks1} (comprising neddylated Cullin1–Rbx1 and the substrate receptor complex Skp1–Skp2–Cks1), facilitating structural analysis by chemical cross-linking mass spectrometry (CX-MS) and cryo-electron microscopy (cryo-EM). Our results indicate the presence of multiple distinct conformations of the catalytic module (comprising the RING domain of Rbx1, CDC34–Ub, and the acceptor ubiquitin) within the di- and tetraubiquitination complexes, while the conformation of the Cullin1–Skp1–Skp2–Cks1 subunit remains unchanged. In conclusion, this work expands the toolkit available for chemical trapping strategies and provides advanced insights into CRL-catalyzed substrate polyubiquitination.