Fig. 3: Heterochirality affects caspase-mediated cleavage of target proteins.

Caspases are unable to cleave target sequences containing d-amino acid(s) as determined by chiral-specific DEVDase assays. a In vitro chiral-specific DEVDase assay (in vitro ChiCAS). Shown are the Ac-DEVD-AMC fluorogenic substrate consisting of all-l-AAs, substrates incorporating a single d-AA (Ac-DEVd-AMC), iso-l-AA (Ac-DEVβD-AMC), iso-d-AA (Ac-DEVβd-AMC) or consisting of all-d-AAs (Ac-devd-AMC). Letters in lower case denote d-AAs or iso-d-AAs. b Assay for substrate specificity of purified recombinant human Caspase-3 activity shows that the cleavage activity is stereoselective. Activity was followed in presence of the homochiral substrate and various heterochiral substrates: Ac-DEVD-AMC (homochiral control), Ac-DEVd-AMC, Ac-DEVβD-AMC, Ac-DEVβd-AMC, and Ac-devd-AMC (homochiral peptide with all-d-AAs). Negative controls Ac-DEVA-AMC and Ac-DEVG-AMC were also tested. Relative fluorescence units (R.F.U) were measured. Experiments were done in triplicates. Values are presented as average ± standard deviation (S.D.). P-values from two-sided Mann–Whitney U-test are ****p < 0.0001. Source data are provided as a Source Data file. c Distance profiles between Caspase-3 catalytic site and the peptide cleavage site computed from molecular dynamics (MD) simulations showing that the d-G peptide bond in DEVdG completely dissociates from the Cys163–His121 dyad making the heterochiral sequence inaccessible to nucleophilic attack from the protease enzyme. Source data files of distance profiles has been uploaded to 10.5281/zenodo.7199808. d The Caspase-3 binding pocket hosts the Cys163–His121 catalytic dyad that cleaves the D-G peptide bond of DEVDG. e Predictive models from MD simulations of DEVDG and DEVdG-bound Caspase-3 showing that the Cys163–His121 catalytic dyad remains inaccessible to DEVdG. Source data files of coordinates, topology and trajectory of the MD simulation are uploaded to https://doi.org/10.5281/zenodo.7199808.