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Title: Reprogramming caspase-7 specificity by regio-specific mutations and selection provides alternate solutions for substrate recognition

Abstract

The ability to routinely engineer protease specificity can allow us to better understand and modulate their biology for expanded therapeutic and industrial applications. In this paper, 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,more » 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. Finally, this approach to specificity reprogramming should also be generalizable across a wide range of proteases.« less

Authors:
 [1];  [1];  [1];  [2];  [2];  [1]
  1. Univ. of Massachusetts, Amherst, MA (United States)
  2. Univ. of California, San Francisco, CA (United States)
Publication Date:
Research Org.:
Univ. of Massachusetts, Amherst, MA (United States)
Sponsoring Org.:
USDOE
OSTI Identifier:
1345036
Grant/Contract Number:
AC02-98CH10886; AC02-06CH11357
Resource Type:
Journal Article: Accepted Manuscript
Journal Name:
ACS Chemical Biology
Additional Journal Information:
Journal Volume: 11; Journal Issue: 6; Journal ID: ISSN 1554-8929
Publisher:
American Chemical Society (ACS)
Country of Publication:
United States
Language:
English
Subject:
59 BASIC BIOLOGICAL SCIENCES; 37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CHEMISTRY; apoptosis; conformational change; cysteine protease; caspase reporter; GFP; selection; flow cytometry; exosite; substrate binding; specificity; directed evolution; protease/protein engineering

Citation Formats

Hill, Maureen E., MacPherson, Derek J., Wu, Peng, Julien, Olivier, Wells, James A., and Hardy, Jeanne A. Reprogramming caspase-7 specificity by regio-specific mutations and selection provides alternate solutions for substrate recognition. United States: N. p., 2016. Web. doi:10.1021/acschembio.5b00971.
Hill, Maureen E., MacPherson, Derek J., Wu, Peng, Julien, Olivier, Wells, James A., & Hardy, Jeanne A. Reprogramming caspase-7 specificity by regio-specific mutations and selection provides alternate solutions for substrate recognition. United States. doi:10.1021/acschembio.5b00971.
Hill, Maureen E., MacPherson, Derek J., Wu, Peng, Julien, Olivier, Wells, James A., and Hardy, Jeanne A. 2016. "Reprogramming caspase-7 specificity by regio-specific mutations and selection provides alternate solutions for substrate recognition". United States. doi:10.1021/acschembio.5b00971. https://www.osti.gov/servlets/purl/1345036.
@article{osti_1345036,
title = {Reprogramming caspase-7 specificity by regio-specific mutations and selection provides alternate solutions for substrate recognition},
author = {Hill, Maureen E. and MacPherson, Derek J. and Wu, Peng and Julien, Olivier and Wells, James A. and Hardy, Jeanne A.},
abstractNote = {The ability to routinely engineer protease specificity can allow us to better understand and modulate their biology for expanded therapeutic and industrial applications. In this paper, 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. Finally, this approach to specificity reprogramming should also be generalizable across a wide range of proteases.},
doi = {10.1021/acschembio.5b00971},
journal = {ACS Chemical Biology},
number = 6,
volume = 11,
place = {United States},
year = 2016,
month = 3
}

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  • In Escherichia coli, cytotoxic DNA methyl lesions on the N1 position of purines and N3 position of pyrimidines are primarily repaired by the 2-oxoglutarate (2-OG) iron(II) dependent dioxygenase, AlkB. AlkB repairs 1-methyladenine (1-meA) and 3-methylcytosine (3-meC) lesions, but it also repairs 1-methylguanine (1-meG) and 3-methylthymine (3-meT) at a much less efficient rate. How the AlkB enzyme is able to locate and identify methylated bases in ssDNA has remained an open question. We determined the crystal structures of the E. coli AlkB protein holoenzyme and the AlkB-ssDNA complex containing a 1-meG lesion. We coupled this to site-directed mutagenesis of amino acidsmore » in and around the active site, and tested the effects of these mutations on the ability of the protein to bind both damaged and undamaged DNA, as well as catalyze repair of a methylated substrate. A comparison of our substrate-bound AlkB-ssDNA complex with our unliganded holoenzyme reveals conformational changes of residues within the active site that are important for binding damaged bases. Site-directed mutagenesis of these residues reveals novel insight into their roles in DNA damage recognition and repair. Our data support a model that the AlkB protein utilizes at least two distinct conformations in searching and binding methylated bases within DNA: a 'searching' mode and 'repair' mode. Moreover, we are able to functionally separate these modes through mutagenesis of residues that affect one or the other binding state. Finally, our mutagenesis experiments show that amino acid D135 of AlkB participates in both substrate specificity and catalysis.« less
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