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

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.