Exploring alternative catalytic mechanisms of the Cas9 HNH domain

Understanding the reaction mechanism of CRISPR‐associated protein 9 (Cas9) is crucial for the application of programmable gene editing. Despite the availability of the structures of Cas9 in apo‐ and substrate‐bound forms, the catalytically active structure is still unclear. Our first attempt to expl...

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Bibliographic Details
Published in:Proteins, structure, function, and bioinformatics structure, function, and bioinformatics, 2020-02, Vol.88 (2), p.260-264
Main Authors: Zhao, Li Na, Mondal, Dibyendu, Warshel, Arieh
Format: Article
Language:English
Subjects:
Biocatalysis
Cas9
Cas9 HNH domain
Catalytic Domain
catalytic mechanism
CRISPR
CRISPR-Associated Protein 9 - chemistry
CRISPR-Associated Protein 9 - metabolism
CRISPR-Cas Systems
Cryoelectron Microscopy
DNA - chemistry
DNA - metabolism
DNA Cleavage
Endonuclease
Gene Editing
Genetic modification
Magnesium
Molecular Dynamics Simulation
Nucleic Acid Conformation
Protein Domains
Reaction mechanisms
RNA, Guide, CRISPR-Cas Systems - chemistry
RNA, Guide, CRISPR-Cas Systems - metabolism
Substrates
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Summary:Understanding the reaction mechanism of CRISPR‐associated protein 9 (Cas9) is crucial for the application of programmable gene editing. Despite the availability of the structures of Cas9 in apo‐ and substrate‐bound forms, the catalytically active structure is still unclear. Our first attempt to explore the catalytic mechanism of Cas9 HNH domain has been based on the reasonable assumption that we are dealing with the same mechanism as endonuclease VII, including the assumption that the catalytic water is in the first shell of the Mg2+. Trying this mechanism with the cryo‐EM structure forced us to induce significant structural change driven by the movement of K848 (or other positively charged residue) close to the active site to facilitate the proton transfer step. In the present study, we explore a second reaction mechanism where the catalytic water is in the second shell of the Mg2+ and assume that the cryo‐EM structure by itself is a suitable representation of a catalytic‐ready structure. The alternative mechanism indicates that if the active water is from the second shell, then the calculated reaction barrier is lower compared with the corresponding barrier when the water comes from the first shell.
ISSN:0887-3585
1097-0134
DOI:10.1002/prot.25796