Evolution of Specificity and Stability in the Folding Trajectory of Caspase

Abstract

Proteins must fold properly and efficiently to accomplish cellular functions. Over the course of evolution, the energy landscape must be maintained such that the protein folds in the right conformation, however, how protein's energy landscape is maintained or altered is unclear. To study how a protein's energy landscape changed over time, we characterized the folding trajectories of caspase homologs and their common ancestor. Caspases are an ancient class of cysteinyl proteases that control apoptosis, responsible for cell differentiation, and maintain cellular homeostasis in multicellular organisms. The caspase family is an excellent model to study protein evolution because substrate specificity and conformational selection are retained through hundreds of millions of years of evolution. Some regulatory features are ancient, and therefore common, while other features are modern and cluster‐specific. In this study, we performed enzyme kinetic assay using synthetic tetrapeptides and substrate phage display using peptide library on ancestral reconstructed and modern proteins to study the evolution of substrate specificity. In addition, we examined urea‐induced equilibrium unfolding properties to evaluate thermodynamic stability and folding pathways. We found that common ancestor of effector caspases showed promiscuity over substrates (D/L/I/VXXD) which is specified later on in modern proteins in different lineages; caspase‐3 and ‐7 prefers DXXD whereas, caspase‐6 prefers VXXD. On the other hand, equilibrium unfolding result suggests all the human effector caspases unfolds via four‐state process; the native dimer (N2) undergoes an isomerization to a dimeric intermediate (I2) and to a monomeric intermediate (I) before completely unfolded (U). Overall, the data show that the folding landscape was established in the early ancestor (>650 MYA) and enzyme specificity evolved from the common landscape. A discrepancy in the overall ΔG0conf, in the folding process between extant caspases, may be due to subsequent mutations in the subfamily.Support or Funding InformationDepartment of Biology, University of Texas at ArlingtonThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.