Characterization of an alternative BAK-binding site for BH3 peptides

Abstract

Many cellular stresses are transduced into apoptotic signals through modification or upregulation of the BH3-only subfamily of BCL2 proteins. Through direct or indirect mechanisms, these proteins activate BAK and BAX to permeabilize the mitochondrial outer membrane. While the BH3-only proteins BIM, PUMA, and tBID have been confirmed to directly activate BAK through its canonical BH3 binding groove, whether the BH3-only proteins BMF, HRK or BIK can directly activate BAK is less clear. Here we show that BMF and HRK bind and directly activate BAK. Through NMR studies, site-directed mutagenesis, and advanced molecular dynamics simulations, we also find that BAK activation by BMF and possibly HRK involves a previously unrecognized binding groove formed by BAK α4, α6, and α7 helices. Alterations in this groove decrease the ability of BMF and HRK to bind BAK, permeabilize membranes and induce apoptosis, suggesting a potential role for this BH3-binding site in BAK activation.

High‐resolution analysis of the conformational transition of pro‐apoptotic Bak at the lipid membrane

“…In order to obtain a more detailed structural picture of full‐length Bak located at the membrane surface, we used NMR to analyze Bak in its membrane‐attached form. So far, NMR resonance assignments were available of N‐terminally truncated constructs of the Bak soluble domain alone (Moldoveanu et al , 2006; Ye et al , 2020), or in complex with a Bid‐BH3 peptide (Moldoveanu et al , 2013). A detailed comparison with our BakΔTM protein construct harboring an intact N‐terminus revealed markedly altered NMR backbone amide spectra.…”

Apoptosis regulation at the mitochondria membrane level

“…An alternate binding site has also been implicated in Bak activation. Bmf and Hrk, two BH3-only proteins whose specific apoptotic activating mechanisms are unclear, have recently been shown to not only directly bind at the hydrophobic groove of Bak, but can do so at an alternative site to the canonical site [65]. Bmf and Hrk can bind both the canonical α3/α4/α5 groove and the alternative α4/α6/α7 groove, with Bmf showing a mild preference for the non-canonical groove.…”

Structural basis of BAK activation in mitochondrial apoptosis initiation

“…BAK activation is triggered by a transient “hit-and-run”, low-affinity binding interaction of activated BID, BIM, and possibly other BH3-only proteins at the canonical hydrophobic groove of BAK 16 , 18 , 19 , 27 , 28 . A recent report implicates BAK activation by interaction of BH3-only proteins BMF and HRK at an alternative site on the opposite face from the canonical groove 29 . How BH3 binding induces BAK to change conformation and awaken during apoptosis is unclear.…”