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Unique structural and dynamic properties of the HNH nuclease in mesophilic and thermophilic Cas9 revealed by NMR
Author(s) -
Belato Helen,
D’Ordine Alexandra,
Jogl Gerwald,
Lisi George
Publication year - 2022
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.2022.36.s1.l7446
Subject(s) - thermophile , nuclease , cas9 , crispr , molecular dynamics , computational biology , chemistry , biophysics , biology , dna , biochemistry , enzyme , computational chemistry , gene
CRISPR‐Cas9 is a widely utilized biochemical tool with applications in biotechnology and in medicine. Target DNA recognition and nuclease sites of Cas9 are spatially separated yet functionally coupled, indicative of an allosteric crosstalk throughout the enzyme. There are structural and dynamic changes occuring throughout Cas9 that are critical for this signal propagation. However, an understanding of the molecular motions guiding Cas9 and how this influences its mechanism are unknown. In this study we report on the structural and dynamic properties of the HNH domain between the extensively studied Sp Cas9 from Streptococcus pyogenes , and a recently discovered thermostable Cas9 homolog from a thermophilic bacterium Geobacillus stearothermophilus ( Geo Cas9). Here we show that despite sequential and structural conservation, Sp HNH and Geo HNH have different dynamic profiles. We found the intrinsic dynamics of Sp HNH to be on the slow (ms) timescale, while Geo HNH is highly influenced by fast (ps‐ns) timescale dynamics. Furthermore, we found that when the residue with the highest flexibility on the ps‐ns timescale is mutated in Geo HNH, its protein solubility, thermal stability, and dynamic profile are all drastically distorted. Our results demonstrate how there are mechanistic differences between the HNH domain of mesophilic and thermophilic Cas9 species. Our results also highlight how these fast timescale motions that regulate Geo HNH play a role in giving Geo Cas9 its thermophilic characteristic.