Altering Caspase Specificity Using an Intracellular Directed Evolution Approach

Abstract

Caspases are among the most specific of proteases, making them ideal targets for engineering new specificity and developing new protease‐based biotherapeutics. However, conventional high‐throughput methods for protein evolution are not amenable to caspases as they are multi‐chain, and intracellular. Increasing the complexity, active sites of caspases are highly flexible to allow for accommodation of various substrates, making a rational design approach difficult To overcome these challenges, we developed a method for evolving function in cytosolic proteases, based on our novel genetically‐encoded caspase activatable‐GFP reporter (CA‐GFP), which is activated from a dark to a fluorescent state by proteolysis.Our first application of this technology, we engineered caspase‐7, which cleaves the amino acid sequence DEVD to recognize and cleave the new sequence VEID, recognized by caspase‐6. Saturation mutagenesis at key residues within the active site of the enzyme allowed us to sort for variants with altered specificity by flow cytometry. Variants able to cleave the target sequence were purified, then characterized using traditional Michaelis‐Menten kinetics, protein substrate assays and x‐ray crystallography. We identified several variants that displayed kinetic activity and cleavage patterns similar to the wild type caspase‐6. X‐ray crystal structures of the evolved variants bound to the casp‐6 and ‐7 cognate substrates VEID, and DEVD reveal how the mutations affect the activity of the enzyme, which could not be rationally predicted, emphasizing the strength of this approach. Utilizing this method we are able to reengineer cytosolic proteases to cleave novel targets and pave the way for biotherapeutic applications.This work is supported in part by NIH GM080532.