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. Characterisation 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. More routine biophysical methods with broader applicability to varied samples is desirable 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 (CRL2 VHL ) is the foundational example. Native mass spectrometry (nMS) can be used to characterise protein complexes but has not previously been used to characterise a full E3 or any E3-E2 interactions. Here, we show that CRL2 VHL is amenable to characterisation by nMS and its interactions with the other protein components integral to the targeted protein degradation mechanism can be observed. Specifically, we characterise binary, ternary and higher order complexes that comprise CRL2 VHL , including the multiprotein systems of POI-PROTAC-CRL2 VHL , CRL2 VHL -E2-Ub, and POI-PROTAC-CRL2 VHL -E2-Ub, all of which are essential in facilitating productive POI ubiquitination and degradation. We benchmarked the nMS with two POI examples (BRD4 BD2 and KRAS) with their respective PROTACs (MZ1 and ACBI3) and were able to observe all relevant complexes across both systems. We anticipate that our findings will open avenues for nMS to integrate as an alternative experimental method enabling scalable characterisation of the intricate high mass multiprotein interactions central to the PROTAC mechanism of action.