Structural Characterization of Degrader-Induced Ternary Complexes Using Hydrogen-Deuterium Exchange Mass Spectrometry...

Area of Science:

• Biochemistry and Structural Biology
• Chemical Biology
• Drug Discovery and Development

Background:

• Targeted protein degradation (TPD) utilizes the ubiquitin-proteasome system to selectively degrade proteins.
• Heterobifunctional degraders recruit target proteins to E3 ligases, forming a ternary complex (target-degrader-ligase).
• Understanding ternary complex geometry is vital for optimizing degrader efficacy, selectivity, and design.

Purpose of the Study:

• To employ hydrogen-deuterium exchange mass spectrometry (HDX-MS) for identifying degrader-induced protein-protein interfaces.
• To construct three-dimensional models of ternary complexes using HDX-MS data and constrained protein docking.
• To characterize the solution structures of ternary complexes formed by CRBN, BRD4's first bromodomain, and two distinct degraders (CFT-1297 and dBET6).

Main Methods:

• Utilized hydrogen-deuterium exchange mass spectrometry (HDX-MS) to probe protein-protein interactions within ternary complexes.
• Applied constrained protein docking, guided by HDX-MS data, to generate three-dimensional structural models.
• Investigated the impact of two different degraders (CFT-1297 and dBET6) on complex formation and structure.

Main Results:

• Distinct deuterium uptake patterns were observed for ternary complexes formed with CFT-1297 and dBET6, indicating different solution structures.
• CFT-1297 demonstrated positive cooperativity, revealing a degrader-induced interface through altered deuterium uptake profiles.
• HDX-MS-constrained docking models for CFT-1297 complexes differed significantly from existing crystal structures, suggesting conformational flexibility.

Conclusions:

• HDX-MS provides rapid structural insights into degrader-induced protein-protein interfaces in solution.
• Degrader ternary complexes exhibit substantial conformational flexibility, and biologically relevant interactions may not involve maximal surface contact.
• Future degrader design should consider linker conformation uncertainty and employ scoring functions adaptable to interfaces lacking evolved complementarity.