Interface Architecture of a VHL-PROTAC Complex with and without Cullin-2

Proteolysis Targeting Chimeras (PROTACs) are bispecific molecules that link a target protein to an E3 ligase, leading to ubiquitination and subsequent degradation. Their efficacy depends on their ability to form ternary complexes capable of being ubiquitinated, which are influenced by protein-protein interactions. Native mass spectrometry combined with surface‑induced dissociation (SID) is a sensitive technique for rapidly assessing protein structures, including stoichiometry and interfacial strengths. Beyond these measurements, native mass spectrometry can also capture a variety of conformational states in the gas phase, reflecting the intrinsic flexibility of many protein assemblies. This ability to resolve structural heterogeneity and transient subpopulations provides complementary insights not as readily accessible through crystallography, cryo‑EM, or other ensemble‑averaging assays. By coupling native mass spectrometry with surface induced dissociation, topological features, specifically relative interfacial strengths and subcomplex arrangements, were probed with and without the scaffold protein Cullin-2 added to a PROTAC-mediated ternary complex. PROTAC-mediated complexes yield rich SID fragmentation into several subcomplexes. Collectively, the SID fragmentation data allow for a rapid and sensitive way to probe PROTAC-mediated protein-protein interactions. Although PROTACs facilitate strong, noncovalent interactions between the target protein and the E3 ligase, the addition of Cullin-2 conformationally locks the E3 ligase complex. This results in a pronounced reduction in fragmentation and offers critical insight into the hierarchical connectivity of the tertiary complex.