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 SpCas9 from Streptococcus pyogenes, and a recently discovered thermostable Cas9 homolog from a thermophilic bacterium Geobacillus stearothermophilus (GeoCas9). Here we show that despite sequential and structural conservation, SpHNH and GeoHNH have different dynamic profiles. We found the intrinsic dynamics of SpHNH to be on the slow (ms) timescale, while GeoHNH 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 GeoHNH, 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 GeoHNH play a role in giving GeoCas9 its thermophilic characteristic.
