Mapping the BH3 Binding Interface of Bcl-xL, Bcl-2, and Mcl-1 Using Split-Luciferase Reassembly

The recognition of helical BH3 domains by Bcl-2 homology (BH) receptors plays a central role in apoptosis. The residues that determine specificity or promiscuity in this interactome are difficult to predict from structural and computational data. Using a cell free split-luciferase system, we have generated a 276 pairwise interaction map for 12 alanine mutations at the binding interface for three receptors, Bcl-xL, Bcl-2, and Mcl-1, and interrogated them against BH3 helices derived from Bad, Bak, Bid, Bik, Bim, Bmf, Hrk, and Puma. This panel, in conjunction with previous structural and functional studies, starts to provide a more comprehensive portrait of this interactome, explains promiscuity, and uncovers surprising details; for example, the Bcl-xL R139A mutation disrupts binding to all helices but the Bad-BH3 peptide, and Mcl-1 binding is particularly perturbed by only four mutations of the 12 tested (V220A, N260A, R263A, and F319A), while Bcl-xL and Bcl-2 have a more diverse set of important residues depending on the bound helix.