Native Mass Spectrometry Analysis of Cullin-RING Ubiquitin E3 Ligase Complexes in the Context of Targeted Protein Degradation.

The binding of PROTACs to their partner ubiquitin E3 ligase (E3) and a protein of interest (POI) is critical for PROTAC development and validation. Characterization of PROTAC complexes by cryo-electron microscopy and X-ray crystallography is not always feasible, especially where species may be transient and protein structures may not resolve due to flexible domains or intrinsically disordered regions or where the air-water interface poses risks such as dissociation. More routine biophysical methods with broader applicability to varied samples are essential to support the rapidly expanding targeted protein degradation field. The majority of PROTACs in development and in the clinic act through a Cullin-RING E3 ligase (CRL), of which the pentameric von Hippel-Lindau (VHL) Cullin 2 RING E3 complex (CRL2VHL) is the premier example. Native mass spectrometry (nMS) can be used to characterize protein complexes but has not previously been used to characterize any full E3 or any E3-E2 interactions. Here, we show for the first time that CRL2VHL (a full E3) is amenable to characterization by nMS and, notably, that all its key interactions with the other protein components integral to the targeted protein degradation mechanism can be observed. Specifically, we characterize binary, ternary, and higher-order complexes that comprise CRL2VHL, including the multiprotein systems of POI-PROTAC-CRL2VHL, CRL2VHL-E2-Ub, and POI-PROTAC-CRL2VHL-E2-Ub, all of which are essential in facilitating productive POI ubiquitination and degradation. We benchmarked the nMS with two POI examples (BRD4BD2 and KRAS) with their respective PROTACs (MZ1 and ACBI3) and were able to observe all of the relevant complexes across both systems. We anticipate that our findings will open avenues for nMS to integrate as an alternative experimental method enabling scalable characterization of the intricate high-mass multiprotein interactions central to biological mechanisms of action.