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Engineered Caspases with Altered Specificities Enable Identification of Exosites
Author(s) -
Hardy Jeanne A,
Hill Maureen,
MacPherson Derek J,
Wu Peng,
Julien Olivier,
Wells James A
Publication year - 2016
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.30.1_supplement.842.10
Subject(s) - caspase , active site , proteases , cleavage (geology) , biochemistry , binding site , caspase 3 , chemistry , caspase 7 , enzyme , biology , apoptosis , programmed cell death , paleontology , fracture (geology)
Exosites for substrate binding are promising for development of inhibitors and activators with novel mechanisms of actions. Despite their potential, to date there is no systematic way to identify exosites on enzymes. We have developed a method for identifying exosites, and applied it to an important class of proteases, the caspases. To identify exosites, we built a version of caspase‐7 that maintained all exosites, but that has the active‐site specificity of caspase‐6. This new caspase allows us to categorize substrates as being active‐site or exosite driven. Simple substitution of the critical active‐site residues from caspase‐6 dramatically diminished caspase‐7 activity, but did not alter specificity, indicating that a more holistic evolutionary procedure would be needed to achieved the desired hybrid caspase. Using our GFP‐based reporter of caspase activity (CA‐GFP), we implemented a flow‐cytometry based directed evolution screen for caspase‐7 variants with caspase‐6 cleavage patterns. The caspase‐7 variants with the most strongly altered specificity displayed kinetic values similar to wild‐type caspase‐6 and mimicked caspase‐6 cleavage patterns with protein substrates. Our seven crystal structures of evolved caspases bound to either DEVD or VEID substrates allowed us to observe how the new residues in the active site contributed to the change in specificity by altering the conformation of a critical active site loop. Using N‐terminomics on the entire human proteome, we observed that the substrate specificity of the evolved caspase was superimposible with caspase‐6 and unlike the parent scaffold, caspase‐7, suggesting that our directed evolution was successful. This allowed us to identify Lamin A/C as an exosite dependent substrate. Ultimately we hope that this approach will identify exosites for recognition of just a subset of key regulatory or disease‐causing substrates, which will lead to more specific small‐molecule modulators of caspase function. Support or Funding Information This work was supported by the National Institutes of Health (GM80532) to JAH and (GM081051) to JAW.