Native Mass Spectrometry of Complexes Formed by Molecular Glues Reveals Stoichiometric Rearrangement of E3 Ligases

Abstract In this application of native mass spectrometry (nMS) to investigate complexes formed by molecular glues (MGs), we have demonstrated its efficiency in delineating stoichiometric rearrangements of E3 ligases that occur during targeted protein degradation (TPD). MGs stabilise interactions between an E3 ligase and a protein of interest (POI) targeted for degradation, and these ternary interactions are challenging to characterise. We have shown that nMS can unambiguously identify complexes formed between the CRBN:DDB1 E3 ligase and the POI GSPT1 upon the addition of lenalidomide, pomalidomide or thalidomide. Ternary complex formation was also identified involving the DCAF15:DDA1:DDB1 E3 ligase in the presence of MG (E7820 or indisulam) and POI RBM39. Moreover, we uncovered that the DCAF15:DDA1:DDB1 E3 ligase self-associates into dimers and trimers when analysed alone at low salt concentrations (100 mM ammonium acetate) which dissociate into single copies of the complex at higher salt concentrations (500 mM ammonium acetate), or upon the addition of MG and POI, forming a 1:1:1 ternary complex. This work demonstrates the strength of nMS in TPD research, reveals novel binding mechanisms of the DCAF15 E3 ligase, and highlights the potential effect of salt concentrations on protein complexes during structural analysis.