Identification of an activation site in Bak and mitochondrial Bax triggered by antibodies

Sweta Iyer; Khatira Anwari; Amber E Alsop; Wai Shan Yuen; David C S Huang; John Carroll; Nicholas A Smith; Brian J Smith; Grant Dewson; Ruth M Kluck

doi:10.1038/ncomms11734

Identification of an activation site in Bak and mitochondrial Bax triggered by antibodies

Nat Commun. 2016 May 24:7:11734. doi: 10.1038/ncomms11734.

Authors

Affiliations

¹ Molecular Genetics of Cancer Division, The Walter and Eliza Hall Institute of Medical Research, Victoria 3052, Australia.
² Development and Stem Cells Program, Monash Biomedicine Discovery Institute and Department of Anatomy and Developmental Biology, Monash University, Melbourne, Victoria 3800, Australia.
³ Cancer and Haematology Division, The Walter and Eliza Hall Institute of Medical Research, Victoria 3052, Australia.
⁴ Department of Chemistry and Physics, La Trobe Institute for Molecular Sciences, La Trobe University, Victoria 3086, Australia.
⁵ Cell Signalling and Cell Death Division, The Walter and Eliza Hall Institute of Medical Research, Victoria 3052, Australia.

Abstract

During apoptosis, Bak and Bax are activated by BH3-only proteins binding to the α2-α5 hydrophobic groove; Bax is also activated via a rear pocket. Here we report that antibodies can directly activate Bak and mitochondrial Bax by binding to the α1-α2 loop. A monoclonal antibody (clone 7D10) binds close to α1 in non-activated Bak to induce conformational change, oligomerization, and cytochrome c release. Anti-FLAG antibodies also activate Bak containing a FLAG epitope close to α1. An antibody (clone 3C10) to the Bax α1-α2 loop activates mitochondrial Bax, but blocks translocation of cytosolic Bax. Tethers within Bak show that 7D10 binding directly extricates α1; a structural model of the 7D10 Fab bound to Bak reveals the formation of a cavity under α1. Our identification of the α1-α2 loop as an activation site in Bak paves the way to develop intrabodies or small molecules that directly and selectively regulate these proteins.

Publication types

Research Support, Non-U.S. Gov't

MeSH terms

Animals
Antibodies, Monoclonal / metabolism*
Apoptosis / physiology*
Cells, Cultured
Cytochromes c / metabolism
Cytosol / metabolism
Epitope Mapping / methods
Epitopes / metabolism*
Female
Fibroblasts
Gene Knockout Techniques
Humans
Mice
Mice, Inbred C57BL
Mitochondria / metabolism
Molecular Docking Simulation
Molecular Dynamics Simulation
Mutagenesis, Site-Directed
Oocytes
Protein Binding / physiology
Protein Conformation, alpha-Helical
Protein Multimerization / physiology
bcl-2 Homologous Antagonist-Killer Protein / chemistry
bcl-2 Homologous Antagonist-Killer Protein / genetics
bcl-2 Homologous Antagonist-Killer Protein / metabolism*
bcl-2-Associated X Protein / chemistry
bcl-2-Associated X Protein / genetics
bcl-2-Associated X Protein / metabolism*

Substances

Antibodies, Monoclonal
BAK1 protein, human
BAX protein, human
Bak1 protein, mouse
Bax protein, mouse
Epitopes
bcl-2 Homologous Antagonist-Killer Protein
bcl-2-Associated X Protein
Cytochromes c

Grants and funding

G1001705/MRC_/Medical Research Council/United Kingdom