Disruption of Bcr-Abl Coiled Coil Oligomerization by Design

• DOI: 10.1074/jbc.m111.264903
• Publication Date: 2011-06-10T08:22:44Z
• Authors: Andrew S. Dixon; Scott S. Pendley; Benjamin J. Bruno; David W. Woessner; Adrian A. Shimpi; Thomas E. Cheatham; Carol S. Lim
• Keywords: Models, Molecular; 0301 basic medicine; Blotting, Western; Fusion Proteins, bcr-abl; Hydrogen Bonding; 03 medical and health sciences; Biopolymers; Microscopy, Fluorescence; Mutagenesis; Two-Hybrid System Techniques; COS Cells; Chlorocebus aethiops; Animals; Humans; Thermodynamics; Computer Simulation; K562 Cells; Cell Proliferation; Plasmids; Protein Binding

Oligomerization is an important regulatory mechanism for many proteins, including oncoproteins and other pathogenic proteins. The oncoprotein Bcr-Abl relies on oligomerization via its coiled coil domain for its kinase activity, suggesting that a designed coiled coil domain with enhanced binding to Bcr-Abl and reduced self-oligomerization would be therapeutically useful. Key mutations in the coiled coil domain of Bcr-Abl were identified that reduce homo-oligomerization through intermolecular charge-charge repulsion yet increase interaction with the Bcr-Abl coiled coil through additional salt bridges, resulting in an enhanced ability to disrupt the oligomeric state of Bcr-Abl. The mutations were modeled computationally to optimize the design. Assays performed in vitro confirmed the validity and functionality of the optimal mutations, which were found to exhibit reduced homo-oligomerization and increased binding to the Bcr-Abl coiled coil domain. Introduction of the mutant coiled coil into K562 cells resulted in decreased phosphorylation of Bcr-Abl, reduced cell proliferation, and increased caspase-3/7 activity and DNA segmentation. Importantly, the mutant coiled coil domain was more efficacious than the wild type in all experiments performed. The improved inhibition of Bcr-Abl through oligomeric disruption resulting from this modified coiled coil domain represents a viable alternative to small molecule inhibitors for therapeutic intervention.