Structural Characterization of BCL-2 Family Protein Interactions Using Photoreactive Stapled Peptides and Mass Spectrometry

Abstract

Recent improvements in mass spectrometry instrumentation have stimulated the fusion of this technology with protein crosslinking to advance the structural proteomics field. However, analysis of complex datasets from crosslinking experiments remains a bottleneck. The majority of crosslinking studies for structural characterization of protein- protein interactions have been conducted with reagents specific for discrete amino acids. While this approach simplifies data analysis, the requirement for specific functionalities to be present at the interaction interface limits resolution. Herein, we report the application of stapled peptides for the development of photoaffinity reagents for mass spectrometric characterization of BCL-2 family protein interactions. To validate this approach, we synthesized photoreactive stabilized alpha-helices of BH3 domains (pSAHBs) incorporating a benzophenone containing amino acid, and demonstrated that the photo crosslinking specificity of these reagents paralleled the interaction specificity of the native proteins. We show that the standard SEQUEST algorithm is effective at identifying specific amino acids crosslinked by pSAHBs, and that this information can be used to create distance restraints for characterizing interaction interfaces by in silico docking. The pSAHB approach is applied to characterize previously elusive activating interactions between BH3 domains and the proapoptotic proteins BAX and BAK. We demonstrate that full-length BAK requires a direct activation stimulus, and that this involves interaction at a canonical surface groove at the C-terminal face of BAK. We confirmed that initiation of direct BAX activation occurs at a geographically distinct site at the N-terminal face of BAX, but further find that induced release of its C-terminus from the canonical groove exposes these residues for secondary BH3 interaction. These data suggest that BAX may be subject to a two-step activation mechanism within distinct cytosolic and mitochondrial compartments. Finally, we report the structural characterization of an interaction between BAD and glucokinase, the first description of a BH3 domain interaction with a non-BCL-2 family member. We identify the active site region of glucokinase as the BAD interaction site, establishing this region as a novel target for development of glucokinase activators. We conclude that the pSAHB approach represents a rapid and powerful approach to protein interaction site identification that complements conventional structural biology techniques.