A Comparison of Two Strategies for Affinity Maturation of a BH3 Peptide toward Pro-Survival Bcl-2 Proteins

作者：Zhang Siyan; Long Angel; Link A James^*

来源：ACS Synthetic Biology, 2012, 1(3): 89-98.

DOI：10.1021/sb200002m

The Bd-2 family of proteins regulates apoptosis at the level of mitochondrial permeabilization. Pro-death members of the family, including Bak and Box, initiate apoptosis, whereas pro-survival members such as Bd-x(L), and Mcl-1 antagonize the function of Bak and Bax via heterodimeric interactions. These heterodimeric interactions are primarily mediated by the binding of the helical amphipathic BH3 domain from a pro-death protein to a hydrophobic cleft on the surface of the pro-survival protein. Since high levels of pro-survival Bcl-2 proteins are present in many cancers, peptides corresponding to pro-death BH3 domains hold promise as therapeutics. Here we apply a high-throughput flow cytometry assay to engineer the Bak BH3 domain for improved affinity toward the pro-survival proteins Bcl-x(L), and Mcl-1. Two strategies, engineering the hydrophobic face of the Bak BH3 peptide and increasing its overall helicity, are successful in identifying Bak BH3 variants with improved affinity to Bcl-x(L), and Mcl-1. Hydrophobic face engineering of the Bak BH3 peptide led to variants with up to a 15-fold increase in affinity for Bcl-x(L), and increased specificity toward Bcl-x(L). Engineering of the helicity of Bak BH3 led to modest (3- to 4-fold) improvements in affinity with retention of promiscuous binding to all pro-survival proteins. HeLa cell killing studies demonstrate that the affinity matured Bak BH3 variants retain their expected biological function.

• 出版日期2012-3