Resurrection of ancestral effector caspases identifies novel networks for evolution of substrate specificity

Abstract

Apoptotic caspases evolved with metazoans more than 950 million years ago (MYA), and a series of gene duplications resulted in two subfamilies consisting of initiator and effector caspases. The effector caspase genes (caspases-3, -6, and -7) were subsequently fixed into the Chordata phylum more than 650 MYA when the gene for a common ancestor (CA) duplicated, and the three effector caspases have persisted throughout mammalian evolution. All caspases require an aspartate residue at the P1 position of substrates, so each caspase evolved discrete cellular roles through changes in substrate recognition at the P4 position combined with allosteric regulation. We examined the evolution of substrate specificity in caspase-6, which prefers valine at the P4 residue, compared to caspases-3 and -7, which prefer aspartate, by reconstructing the CA of effector caspases (AncCP-Ef1) and the CA of caspase-6 (AncCP-6An). We show that AncCP-Ef1 is a promiscuous enzyme with little distinction between Asp, Val, or Leu at P4. The specificity of caspase-6 was defined early in its evolution, where AncCP-6An demonstrates preference for Val over Asp at P4. Structures of AncCP-Ef1 and of AncCP-6An show a network of charged amino acids near the S4 pocket that, when combined with repositioning a flexible active site loop, resulted in a more hydrophobic binding pocket in AncCP-6An. The ancestral protein reconstructions show that the caspasehemoglobinase fold has been conserved for over 650 million years and that only three substitutions in the scaffold are necessary to shift substrate selection toward Val over Asp.

Remodeling hydrogen bond interactions results in relaxed specificity of Caspase-3

“…Reconstructions of ancestral effector caspases showed that the polar glutamate in the caspase-6 lineage is moved away from the S4 subsite by hydrogen bonding to a network of amino acids on helices-4 and -5, as described above (11). The interactions result in rotation of a hydrophobic group on active site loop 4 (L4), called GP9-V01, into the S4 subsite ( Figure 1C) (11). The charged network on helices 4 and 5, observed in caspase-6, is incomplete in caspases-3 and -7, and the hydrophobic GP9-V01 is substituted with glutamate in the DxxDases ( Figure 1D).…”

“…In contrast, CP162 is conserved in caspases-3 and -7 (DxxDases) as asparagine (Figure 1, panels B, D and Figure 2E). Reconstructions of ancestral effector caspases showed that the polar glutamate in the caspase-6 lineage is moved away from the S4 subsite by hydrogen bonding to a network of amino acids on helices-4 and -5, as described above (11). The interactions result in rotation of a hydrophobic group on active site loop 4 (L4), called GP9-V01, into the S4 subsite ( Figure 1C) (11).…”

“…We have shown that hydrogen bonding is an important determinant for selection of the P4 residue of caspase substrates and that interactions outside of the S4 binding pocket contribute to the selection of the P4 residue. In the conformational landscape of caspase-6, a network of charged or polar amino acids moves CP-E162 away from the S4 pocket, and rotation of GP9-V01 into the pocket increases hydrophobicity and thus preference for P4 valine in the substrate (11). In the context of the caspase-3 conformational landscape, however, nature did not use this strategy to change selection toward P4 aspartate.…”

“…Caspase subfamilies evolved from a common ancestor that diverged into multiple caspases more than 700 million years ago (10). We showed recently that the common ancestor of effector caspases-3, -6, and -7 was a promiscuous enzyme with little preference for aspartate or valine at the P4 site (11). In addition, specificity for valine evolved early in the caspase-6 lineage due to introduction of a network of charged amino acids near the S4 subsite ( Figure 1C).…”

Caspases from scleractinian coral show unique regulatory features

“…Enzyme activity was determined in a buffer of 150 m m Tris-HCl, pH 7.5, 50 m m NaCl, 10 m m DTT, 1% sucrose, 0.1% CHAPS (assay buffer) at 25°C, as described previously ( 57 , 58 ). The total reaction volume was 200 μl, and the final enzyme concentration was 10 n m .…”