Mapping the BH3 binding interface of Bcl-x<inf>L</inf>, Bcl-2, and Mcl-1 using split-luciferase reassembly

Sean T. Campbell, Kevin J. Carlson, Carl J. Buchholz, Mark R. Helmers, Indraneel Ghosh

Research output: Contribution to journalArticle

4 Citations (Scopus)

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-x<inf>L</inf>, 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-x<inf>L</inf> 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-x<inf>L</inf> and Bcl-2 have a more diverse set of important residues depending on the bound helix.

Original languageEnglish (US)
Pages (from-to)2632-2643
Number of pages12
JournalBiochemistry
Volume54
Issue number16
DOIs
StatePublished - Apr 28 2015

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Luciferases
Alanine
Apoptosis
Mutation
Puma
Bax protein (53-86)

ASJC Scopus subject areas

  • Biochemistry
  • Medicine(all)

Cite this

Mapping the BH3 binding interface of Bcl-x<inf>L</inf>, Bcl-2, and Mcl-1 using split-luciferase reassembly. / Campbell, Sean T.; Carlson, Kevin J.; Buchholz, Carl J.; Helmers, Mark R.; Ghosh, Indraneel.

In: Biochemistry, Vol. 54, No. 16, 28.04.2015, p. 2632-2643.

Research output: Contribution to journalArticle

Campbell, Sean T. ; Carlson, Kevin J. ; Buchholz, Carl J. ; Helmers, Mark R. ; Ghosh, Indraneel. / Mapping the BH3 binding interface of Bcl-x<inf>L</inf>, Bcl-2, and Mcl-1 using split-luciferase reassembly. In: Biochemistry. 2015 ; Vol. 54, No. 16. pp. 2632-2643.
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