14-3-3 protein binding blocks both the nuclear localization sequence and the dimerization interface of caspase-2

D. Kalabova1,2, T. Obsil1,3 a V. Obsilova1

1Department of Structural Biology of Signaling Proteins, Division BIOCEV, Institute of Physiology of the Czech Academy of Sciences, 252 50 Vestec, Czech Republic

22nd Faculty of Medicine, Charles University, 150 06 Prague 5, Czech Republic

3Department of Physical and Macromolecular Chemistry, Faculty of Science, Charles University, 128 43 Prague 2, Czech Republic

veronika.obsilova@fgu.cas.cz

 

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) [1, 2]. Moreover, the caspase-2 region with both 14-3-3-binding motifs also contains the nuclear localization sequence (NLS), thus suggesting that 14-3-3 binding may regulate the subcellular localization of caspase-2. However, the mechanism of 14-3-3-dependent inhibition of C2 activation remains unclear.

We recently reported the structural characterization of the complex between proC2 and 14-3-3 using a combined approach based on small angle X-ray scattering, NMR, chemical cross-linking, fluorescence spectroscopy, hydrogen/deuterium mass spectrometry (HDX-MS) and protein crystallography to determine the molecular basis for 14-3-3-mediated inhibition of C2 activation [3, 4]. Our data revealed 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, the 14-3-3 protein interacts with and masks both the nuclear localization sequence (NLS) 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. Because this masked region of p12 domain is involved in caspase-2 dimerization, 14-3-3 likely inhibits proC2 activation by blocking the dimerization surface of this procaspase.

In conclusion, the structural analysis of the 14-3-3:caspase-2 complex suggested that 14-3-3 protein binding may inhibit caspase-2 activation through interference with caspase-2 oligomerization and/or its nuclear localization by sterically occluding caspase-2 p12 domain as well as NLS, which is bordered by the two phosphorylated 14-3-3-binding motifs of caspase-2. Thus, these results corroborate the hypothesis that 14-3-3 binding is an important regulatory element of caspase-2 activation.

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4.         D. Kalabova, F. Filandr, M. Alblova, O. Petrvalska, M. Horvath, P. Man, T. Obsil T, and V. Obsilova, FEBS J., doi: 10.1111/febs.15215 (2020).

This study was supported by the Czech Science Foundation (Project 20-00058S), the Czech Academy of Sciences (Research Projects RVO: 67985823 of the Institute of Physiology).