title: Mapping the BH3 Binding Interface of Bcl-xL, Bcl-2, and Mcl-1 Using Split-Luciferase Reassembly.
abstract: 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.
authors.affiliation: Department of Chemistry and Biochemistry, University of Arizona, 1306 East University Boulevard, Tucson, Arizona 85721, United States.
authors.forename: Sean T
authors.initials: ST
authors.lastname: Campbell
authors.affiliation: Department of Chemistry and Biochemistry, University of Arizona, 1306 East University Boulevard, Tucson, Arizona 85721, United States.
authors.forename: Kevin J
authors.initials: KJ
authors.lastname: Carlson
authors.affiliation: Department of Chemistry and Biochemistry, University of Arizona, 1306 East University Boulevard, Tucson, Arizona 85721, United States.
authors.forename: Carl J
authors.initials: CJ
authors.lastname: Buchholz
authors.affiliation: Department of Chemistry and Biochemistry, University of Arizona, 1306 East University Boulevard, Tucson, Arizona 85721, United States.
authors.forename: Mark R
authors.initials: MR
authors.lastname: Helmers
authors.affiliation: Department of Chemistry and Biochemistry, University of Arizona, 1306 East University Boulevard, Tucson, Arizona 85721, United States.
authors.forename: Indraneel
authors.initials: I
authors.lastname: Ghosh
mesh_terms: D019943:Amino Acid Substitution; D000818:Animals; D002474:Cell-Free System; D048888:Fireflies; D006801:Humans; D049409:Luciferases, Firefly; D020125:Mutation, Missense; D064549:Myeloid Cell Leukemia Sequence 1 Protein / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D010449:Peptide Mapping; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D051020:bcl-X Protein / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism
chemical_list: C000595707:BCL2 protein, human; C494811:BCL2L1 protein, human; C579095:MCL1 protein, human; D064549:Myeloid Cell Leukemia Sequence 1 Protein; D019253:Proto-Oncogene Proteins c-bcl-2; D051020:bcl-X Protein; C485197:luciferase, Photinus; D049409:Luciferases, Firefly