A double point mutation at residues Ile14 and Val15 of Bcl-2 uncovers a role for the BH4 domain in both protein stability and function

Giovanni Monaco, Rita La Rovere, Spyridoula Karamanou, Kirsten Welkenhuyzen, Hristina Ivanova, Elien Vandermarliere, Marta Di Martile, Donatella Del Bufalo, Humbert De Smedt, Jan B. Parys, Anastassios Economou, Geert Bultynck

Research output: Contribution to journalArticlepeer-review


B-cell lymphoma 2 (Bcl-2) protein is the archetype apoptosis suppressor protein. The N-terminal Bcl-2-homology 4 (BH4) domain of Bcl-2 is required for the antiapoptotic function of this protein at the mitochondria and endoplasmic reticulum (ER). The involvement of the BH4 domain in Bcl-2′s antiapoptotic functions has been proposed based on Gly-based substitutions of the Ile14/Val15 amino acids, two hydrophobic residues located in the center of Bcl-2′s BH4 domain. Following this strategy, we recently showed that a BH4-domain-derived peptide in which Ile14 and Val15 have been replaced by Gly residues, was unable to dampen proapoptotic Ca2+-release events from the ER. Here, we investigated the impact of these mutations on the overall structure, stability, and function of full-length Bcl-2 as a regulator of Ca2+ signaling and cell death. Our results indicate that full-length Bcl-2 Ile14Gly/Val15Gly, in contrast to wild-type Bcl-2, (a) displayed severely reduced structural stability and a shortened protein half-life; (b) failed to interact with Bcl-2-associated X protein (BAX), to inhibit the inositol 1,4,5-trisphosphate receptor (IP3R) and to protect against Ca2+-mediated apoptosis. We conclude that the hydrophobic face of Bcl-2′s BH4 domain (Ile14, Val15) is an important structural regulatory element by affecting protein stability and turnover, thereby likely reducing Bcl-2′s ability to modulate the function of its targets, like IP3R and BAX. Therefore, Bcl-2 structure/function studies require pre-emptive and reliable determination of protein stability upon introduction of point mutations at the level of the BH4 domain.

Original languageEnglish
Pages (from-to)127-145
Number of pages19
JournalFEBS Journal
Issue number1
Publication statusPublished - Jan 1 2018


  • apoptosis
  • Bcl-2
  • Bcl-2-associated X protein
  • Ca signaling
  • hydrophobic core
  • inositol 1,4,5-trisphosphate receptor
  • point mutations
  • protein stability and turnover

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology


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