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 prefer 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 with 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 a 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 caspase-hemoglobinase 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

“…In contrast, CP162 is conserved in caspases-3 and -7 (DxxDases) as asparagine ( Figure 1 B,D and Figure 2 E). 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 [ 12 ]. The interactions result in rotation of a hydrophobic group on active site loop 4 (L4), called GP9-V01, into the S4 subsite ( Figure 1 C) [ 12 ].…”

“…Caspase subfamilies evolved from a common ancestor that diverged into multiple caspases more than 700 million years ago [ 11 ]. 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 [ 12 ]. 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 1 C).…”

Caspases from Scleractinian Coral Show Unique Regulatory Features

“…Enzyme activity was determined in a buffer of 150 mM Tris-HCl, pH 7.5, 50 mM NaCl, 10 mM DTT, 1% sucrose, 0.1% CHAPS (assay buffer) at 25 °C, as previously described (35,36).…”

“…The steady-state parameters, KM and kcat, were determined from plots of initial velocity versus substrate concentration. Substrate phage display assays were performed as described (36,37). Briefly, phage libraries consisting of caspase recognition sequences were bound to Ni-NTA resin.…”

Novel Apoptotic Mediators Identified by Conservation of Vertebrate Caspase Targets

“…The same study by the Wells group demonstrated that substitution of P1 glutamate by aspartate actually results in a higher rate of protein cleavage by caspases. The shared ability of caspases-3, -6, and -7 to cleave substrates after glutamate could be explained by their very similar structure, which originates from a single ancestral effector caspase gene [38]. Both P1 aspartate and glutamate appear to be conserved throughout Metazoans in general [13]; however, a taxon-oriented study showed that mammals have a lower incidence of P1 E-cut sites (3%) compared to birds (7%) and ray-finned fish (9%) (Table S3, Figure S1).…”