14-3-3 protein binding blocks the dimerization interface of caspase-2

FEBS J. 2020 Aug;287(16):3494-3510. doi: 10.1111/febs.15215. Epub 2020 Feb 4.


Among all species, caspase-2 (C2) is the most evolutionarily conserved caspase required for effective initiation of apoptosis following death stimuli. C2 is activated through dimerization and autoproteolytic cleavage and inhibited through phosphorylation at Ser139 and Ser164 , within the linker between the caspase recruitment and p19 domains of the zymogen, followed by association with the adaptor protein 14-3-3, which maintains C2 in its immature form procaspase (proC2). However, the mechanism of 14-3-3-dependent inhibition of C2 activation remains unclear. Here, we report the structural characterization of the complex between proC2 and 14-3-3 by hydrogen/deuterium mass spectrometry and protein crystallography to determine the molecular basis for 14-3-3-mediated inhibition of C2 activation. Our data reveal that the 14-3-3 dimer interacts with proC2 not only through ligand-binding grooves but also through other regions outside the central channel, thus explaining the isoform-dependent specificity of 14-3-3 protein binding to proC2 and the substantially higher binding affinity of 14-3-3 protein to proC2 than to the doubly phosphorylated peptide. The formation of the complex between 14-3-3 protein and proC2 does not induce any large conformational change in proC2. Furthermore, 14-3-3 protein interacts with and masks both the nuclear localization sequence and the C-terminal region of the p12 domain of proC2 through transient interactions in which both the p19 and p12 domains of proC2 are not firmly docked onto the surface of 14-3-3. This masked region of p12 domain is involved in C2 dimerization. Therefore, 14-3-3 protein likely inhibits proC2 activation by blocking its dimerization surface. DATABASES: Structural data are available in the Protein Data Bank under the accession numbers 6SAD and 6S9K.

Keywords: 14-3-3 protein; H/D exchange; caspase-2; crystallography; protein-protein interaction.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • 14-3-3 Proteins / chemistry*
  • 14-3-3 Proteins / genetics
  • 14-3-3 Proteins / metabolism
  • Binding Sites / genetics
  • Caspase 2 / chemistry*
  • Caspase 2 / genetics
  • Caspase 2 / metabolism
  • Crystallography, X-Ray
  • Humans
  • Models, Molecular*
  • Mutation
  • Phosphorylation
  • Protein Binding
  • Protein Conformation*
  • Protein Isoforms / genetics
  • Protein Isoforms / metabolism
  • Protein Multimerization*
  • Protein Precursors / chemistry*
  • Protein Precursors / genetics
  • Protein Precursors / metabolism
  • Recombinant Proteins / chemistry
  • Recombinant Proteins / metabolism


  • 14-3-3 Proteins
  • Protein Isoforms
  • Protein Precursors
  • Recombinant Proteins
  • Caspase 2