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

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 ProbeUb2 and Extension ProbeUb4, 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 ProbeUb4, 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 ProbeUb2 and Extension ProbeUb4 formed stable complexes with N8-CRL1Skp1/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.