4ZVQ

Caspase-7 Variant 2 (V2) with reprogrammed substrate specificity due to Y230V/W232M/Q276C substitutions bound to VEID inhibitor.

4ZVQ の概要

• エントリーDOI: 10.2210/pdb4zvq/pdb
• 関連するPDBエントリー: 1F1J 3EDR 4ZVO 4ZVP 4ZVR 4ZVS 4ZVT 4ZVU
• 関連するBIRD辞書のPRD_ID: PRD_000976
• 分子名称: Caspase-7, Peptide ACE-VAL-GLU-ILE-ASA, ... (4 entities in total)
• 機能のキーワード: directed evolution, protease, peptide inhibitor, designed active site specificity, hydrolase-hydrolase inhibitor complex, hydrolase/hydrolase inhibitor
• 由来する生物種: Homo sapiens (Human); 詳細
• タンパク質・核酸の鎖数: 6
• 化学式量合計: 71507.01

構造登録者

Hill, M.E.,MacPherson, D.J.,Hardy, J.A. (登録日: 2015-05-18, 公開日: 2016-04-20, 最終更新日: 2024-11-20)

主引用文献

Hill, M.E.,MacPherson, D.J.,Wu, P.,Julien, O.,Wells, J.A.,Hardy, J.A.
Reprogramming Caspase-7 Specificity by Regio-Specific Mutations and Selection Provides Alternate Solutions for Substrate Recognition.
Acs Chem.Biol., 11:1603-1612, 2016

PubMed Abstract: The ability to routinely engineer protease specificity can allow us to better understand and modulate their biology for expanded therapeutic and industrial applications. Here, we report a new approach based on a caged green fluorescent protein (CA-GFP) reporter that allows for flow-cytometry-based selection in bacteria or other cell types enabling selection of intracellular protease specificity, regardless of the compositional complexity of the protease. Here, we apply this approach to introduce the specificity of caspase-6 into caspase-7, an intracellular cysteine protease important in cellular remodeling and cell death. We found that substitution of substrate-contacting residues from caspase-6 into caspase-7 was ineffective, yielding an inactive enzyme, whereas saturation mutagenesis at these positions and selection by directed evolution produced active caspases. The process produced a number of nonobvious mutations that enabled conversion of the caspase-7 specificity to match caspase-6. The structures of the evolved-specificity caspase-7 (esCasp-7) revealed alternate binding modes for the substrate, including reorganization of an active site loop. Profiling the entire human proteome of esCasp-7 by N-terminomics demonstrated that the global specificity toward natural protein substrates is remarkably similar to that of caspase-6. Because the esCasp-7 maintained the core of caspase-7, we were able to identify a caspase-6 substrate, lamin C, that we predict relies on an exosite for substrate recognition. These reprogrammed proteases may be the first tool built with the express intent of distinguishing exosite dependent or independent substrates. This approach to specificity reprogramming should also be generalizable across a wide range of proteases.

PubMed: 27032039
DOI: 10.1021/acschembio.5b00971

実験手法

X-RAY DIFFRACTION (2.5 Å)