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Updated: May 7, 2026
Author Spotlight: Evaluating Biophysical Assays for Characterizing PROTACS Ternary Complexes
Published on: January 12, 2024
Interface Architecture of a VHL-PROTAC Complex with and without Cullin-2
Evan N Whitford1,2, Joshua D Gilbert3, Marius M Kostelic3,4
1School of Chemistry & Biochemistry, Georgia Institute of Technology, Atlanta, Georgia 30332, United States.
Abstract:
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 for target ubiquitination, which is 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. 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 ternary complexes yield rich SID fragmentation into several subcomplexes. The extensive fragmentation observed for the PROTAC-assembled complex lacking Cullin-2 suggests that this Cullin-free ternary complex is more conformationally flexible, enabling multiple accessible subcomplex topologies. Although PROTACs facilitate strong, noncovalent interactions between the target protein and the E3 ligase, the addition of Cullin-2 reduced the conformational flexibility of the E3 ligase complex. This results in a pronounced reduction in fragmentation and offers critical insight into the hierarchical connectivity of the ternary complex.
Insights
Native mass spectrometry reveals how Proteolysis Targeting Chimeras (PROTACs) form ternary complexes. Adding Cullin-2 to PROTAC complexes reduces flexibility, offering insights into their structural connectivity.
Area of Science:
- Biochemistry
- Structural Biology
- Chemical Biology
Background:
- Proteolysis Targeting Chimeras (PROTACs) are emerging therapeutics that harness the ubiquitin-proteasome system for targeted protein degradation.
- The efficacy of PROTACs relies on the formation of a ternary complex between the target protein, the PROTAC molecule, and an E3 ligase.
- Understanding the structural dynamics and interactions within these ternary complexes is crucial for PROTAC design and optimization.
Purpose of the Study:
- To investigate the structural topology and conformational flexibility of PROTAC-mediated ternary complexes.
- To assess the impact of the scaffold protein Cullin-2 on the stability and fragmentation patterns of these complexes.
- To utilize native mass spectrometry and surface-induced dissociation (SID) for probing interfacial strengths and subcomplex arrangements.
Main Methods:
- Native mass spectrometry was employed to analyze the intact ternary complexes in the gas phase.
- Surface-induced dissociation (SID) was coupled with native mass spectrometry to induce fragmentation and analyze subcomplexes.
- Comparative analysis of fragmentation patterns was performed with and without the addition of Cullin-2.
Main Results:
- PROTAC-mediated ternary complexes without Cullin-2 exhibited extensive fragmentation, indicating significant conformational flexibility and multiple accessible topologies.
- The addition of Cullin-2 to the PROTAC-assembled complex led to a pronounced reduction in fragmentation.
- This reduction in fragmentation suggests that Cullin-2 decreases the conformational flexibility of the E3 ligase complex, impacting ternary complex dynamics.
Conclusions:
- Native mass spectrometry and SID are powerful tools for characterizing the structural heterogeneity and interfacial strengths of PROTAC-mediated ternary complexes.
- Cullin-2 plays a role in modulating the conformational flexibility and hierarchical connectivity of the PROTAC E3 ligase complex.
- These findings provide critical insights into the structural basis of PROTAC action and inform the rational design of more effective PROTACs.
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