7MPZ

HNH Nuclease Domain from G. stearothermophilus Cas9

Summary for 7MPZ

• Entry DOI: 10.2210/pdb7mpz/pdb
• Related: 6J9N 6O56
• Descriptor: CRISPR-associated endonuclease Cas9 (2 entities in total)
• Functional Keywords: nuclease domain, crispr cas9, dna binding protein
• Biological source: Geobacillus stearothermophilus (Bacillus stearothermophilus)
• Total number of polymer chains: 1
• Total formula weight: 18507.87

Authors

D'Ordine, A.M.,Belato, H.B.,Lisi, G.P.,Jogl, G. (deposition date: 2021-05-05, release date: 2021-12-22, Last modification date: 2023-10-18)

Primary citation

Belato, H.B.,D'Ordine, A.M.,Nierzwicki, L.,Arantes, P.R.,Jogl, G.,Palermo, G.,Lisi, G.P.
Structural and dynamic insights into the HNH nuclease of divergent Cas9 species.
J.Struct.Biol., 214:107814-107814, 2021

PubMed Abstract: CRISPR-Cas9 is a widely used biochemical tool with applications in molecular biology and precision medicine. The RNA-guided Cas9 protein uses its HNH endonuclease domain to cleave the DNA strand complementary to its endogenous guide RNA. In this study, novel constructs of HNH from two divergent organisms, G. stearothermophilus (GeoHNH) and S. pyogenes (SpHNH) were engineered from their respective full-length Cas9 proteins. Despite low sequence similarity, the X-ray crystal structures of these constructs reveal that the core of HNH surrounding the active site is conserved. Structure prediction of the full-length GeoCas9 protein using Phyre2 and AlphaFold2 also showed that the crystallographic construct of GeoHNH represents the structure of the domain within the full-length GeoCas9 protein. However, significant differences are observed in the solution dynamics of structurally conserved regions of GeoHNH and SpHNH, the latter of which was shown to use such molecular motions to propagate the DNA cleavage signal. Indeed, molecular simulations show that the intradomain signaling pathways, which drive SpHNH function, are non-specific and poorly formed in GeoHNH. Taken together, these outcomes suggest mechanistic differences between mesophilic and thermophilic Cas9 species.

PubMed: 34871741
DOI: 10.1016/j.jsb.2021.107814

Experimental method

X-RAY DIFFRACTION (2.04 Å)