Caspase-3 binds diverse P4 residues in peptides as revealed by crystallography and structural modeling

Abstract

Caspase-3 recognition of various P4 residues in its numerous protein substrates was investigated by crystallography, kinetics, and calculations on model complexes. Asp is the most frequent P4 residue in peptide substrates, although a wide variety of P4 residues are found in the cellular proteins cleaved by caspase-3. The binding of peptidic inhibitors with hydrophobic P4 residues, or no P4 residue, is illustrated by crystal structures of caspase-3 complexes with Ac-IEPD-Cho, Ac-WEHD-Cho, Ac-YVAD-Cho, and Boc-D(OMe)-Fmk at resolutions of 1.9-2.6 A. The P4 residues formed favorable hydrophobic interactions in two separate hydrophobic regions of the binding site. The side chains of P4 Ile and Tyr form hydrophobic interactions with caspase-3 residues Trp206 and Trp214 within a non-polar pocket of the S4 subsite, while P4 Trp interacts with Phe250 and Phe252 that can also form the S5 subsite. These interactions of hydrophobic P4 residues are distinct from those for polar P4 Asp, which indicates the adaptability of caspase-3 for binding diverse P4 residues. The predicted trends in peptide binding from molecular models had high correlation with experimental values for peptide inhibitors. Analysis of structural models for the binding of 20 different amino acids at P4 in the aldehyde peptide Ac-XEVD-Cho suggested that the majority of hydrophilic P4 residues interact with Phe250, while hydrophobic residues interact with Trp206, Phe250, and Trp214. Overall, the S4 pocket of caspase-3 exhibits flexible adaptation for different residues and the new structures and models, especially for hydrophobic P4 residues, will be helpful for the design of caspase-3 based drugs.

Conformational similarity in the activation of caspase-3 and -7 revealed by the unliganded and inhibited structures of caspase-7

“…However, accommodation of P4 Tyr at the S4 site results in loss of a main chain hydrogen bond between the P4 amide and carbonyl oxygen of Gln276. Similar loss of the equivalent main chain hydrogen bond was observed for P4 Tyr binding at S4 pocket of caspase-3 in our previous study [20]. The inhibition constant of Ac-YVAD-Cho for caspase-7 was 12.4 ± 0.5 lM, which is similar to values reported for weak inhibitors like Ac-WEHD-Cho [16].…”

“…P4 Tyr binds similarly to both enzymes at the S4 subsite, while P4 Trp shows distinct differences in binding to caspase-3 and -7. The aromatic ring of bulky P4 Trp forms favorable interactions with Phe250 and Phe252 that form the S5 pocket in caspase-3 [20]. In contrast, in caspase-7 the side chain of P4 Trp exhibits two different conformations; in one active site Trp binds at the S4 subsite, while the Trp side chain is rotated away from S4 in the second active site.…”

“…It also revealed that the S4 subsite of caspase-7 has two distinct binding sites for polar and aliphatic residues, thereby providing structural evidence for recognition of noncanonical sequences. The bulky aromatic residue tryptophan, which is a preferred P4 residue for inflammatory caspases, was found to be structurally unfavorable as a P4 residue for caspase-3 and -7, although P4 tyrosine was accommodated in caspase-3 [16,20]. The structural effect of P4 tyrosine was not studied for caspase-7 previously.…”

Multi-level structure-based pharmacophore modelling of caspase-3-non-peptide complexes: Extracting essential pharmacophore features and its application to virtual screening

“…aldehydes, vinyl suplhones, epoxides and chloromethyl ketones) to connect nucleophilic cysteinyl thiol group by covalent linkage [21][22][23]. Keeping these inhibitors specificity elements of caspase-3 in mind, various studies involved the replacement of P 1 -P 3 residues by chemical groups which resulted in the development of numerous diverse peptidebased inhibitors [1,6].…”

“…These collective efforts have generated a myriad of caspase-3-non-peptide inhibitor complexes [1,4,6,7,8,20,[24][25][26]. Computational caspase-3 inhibitor design through pharmacophore-based virtual screening utilized relatively less receptor details for strategic design unless are docking approach wherein a single template is explored for analyzing the binding efficiency of diverse molecules [4,27].…”