8F43
HNH Nuclease Domain from G. stearothermophilus Cas9, K597A mutant
Summary for 8F43
| Entry DOI | 10.2210/pdb8f43/pdb |
| Related | 7MPZ |
| Descriptor | CRISPR-associated endonuclease Cas9 (2 entities in total) |
| Functional Keywords | nuclease domain, crispr cas9, dna binding protein, hydrolase |
| Biological source | Geobacillus stearothermophilus |
| Total number of polymer chains | 1 |
| Total formula weight | 13020.69 |
| Authors | D'Ordine, A.M.,Belato, H.B.,Lisi, G.P.,Jogl, G. (deposition date: 2022-11-10, release date: 2022-12-21, Last modification date: 2024-05-01) |
| Primary citation | Belato, H.B.,Norbrun, C.,Luo, J.,Pindi, C.,Sinha, S.,D'Ordine, A.M.,Jogl, G.,Palermo, G.,Lisi, G.P. Disruption of electrostatic contacts in the HNH nuclease from a thermophilic Cas9 rewires allosteric motions and enhances high-temperature DNA cleavage. J.Chem.Phys., 157:225103-225103, 2022 Cited by PubMed Abstract: Allosteric signaling within multidomain proteins is a driver of communication between spatially distant functional sites. Understanding the mechanism of allosteric coupling in large multidomain proteins is the most promising route to achieving spatial and temporal control of the system. The recent explosion of CRISPR-Cas9 applications in molecular biology and medicine has created a need to understand how the atomic level protein dynamics of Cas9, which are the driving force of its allosteric crosstalk, influence its biophysical characteristics. In this study, we used a synergistic approach of nuclear magnetic resonance (NMR) and computation to pinpoint an allosteric hotspot in the HNH domain of the thermostable GeoCas9. We show that mutation of K597 to alanine disrupts a salt-bridge network, which in turn alters the structure, the timescale of allosteric motions, and the thermostability of the GeoHNH domain. This homologous lysine-to-alanine mutation in the extensively studied mesophilic S. pyogenes Cas9 similarly alters the dynamics of the SpHNH domain. We have previously demonstrated that the alteration of allostery via mutations is a source for the specificity enhancement of SpCas9 (eSpCas9). Hence, this may also be true in GeoCas9. PubMed: 36546784DOI: 10.1063/5.0128815 PDB entries with the same primary citation |
| Experimental method | X-RAY DIFFRACTION (1.37 Å) |
Structure validation
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