9B72

Rec2 Domain from G. stearothermophilus Cas9

Summary for 9B72

• Entry DOI: 10.2210/pdb9b72/pdb
• Descriptor: CRISPR-associated endonuclease Cas9 (2 entities in total)
• Functional Keywords: crispr cas9, nucleic acid binding protein, rna binding protein
• Biological source: Geobacillus stearothermophilus
• Total number of polymer chains: 1
• Total formula weight: 25399.81

Authors

D'Ordine, A.M.,Belato, H.B.,Lisi, G.P.,Jogl, G. (deposition date: 2024-03-26, release date: 2025-08-27)

Primary citation

Belato, H.B.,Knight, A.L.,D'Ordine, A.M.,Pindi, C.,Fan, Z.,Luo, J.,Palermo, G.,Jogl, G.,Lisi, G.P.
Structural and dynamic impacts of single-atom disruptions to guide RNA interactions within the recognition lobe of Geobacillus stearothermophilus Cas9.
Elife, 13:-, 2025

PubMed Abstract: The intuitive manipulation of specific amino acids to alter the activity or specificity of CRISPR-Cas9 has been a topic of great interest. As a large multi-domain RNA-guided endonuclease, the intricate molecular crosstalk within the Cas9 protein hinges on its conformational dynamics, but a comprehensive understanding of the extent and timescale of the motions that drive its allosteric function and association with nucleic acids remains elusive. Here, we investigated the structure and multi-timescale molecular motions of the recognition (Rec) lobe of Cas9, a thermophilic Cas9 from . Our results provide new atomic details about the Rec subdomains (Rec1, Rec2) and the full-length domain in solution. Two rationally designed mutants, K267E and R332A, enhanced and redistributed micro-millisecond flexibility throughout Rec, and NMR studies of the interaction between Rec and its guide RNA showed that mutations reduced this affinity and the stability of the ribonucleoprotein complex. Despite measured biophysical differences due to the mutations, DNA cleavage assays reveal no functional differences in on-target activity, and similar specificity. These data suggest that guide RNA interactions can be tuned at the biophysical level in the absence of major functional losses but also raise questions about the underlying mechanism of Cas9, since analogous single-point mutations have significantly impacted on- and off-target DNA editing in mesophilic Cas9. A K267E/R332A double mutant did also did not enhance Cas9 specificity, highlighting the robust tolerance of mutations to the Rec lobe of Cas9 and species-dependent complexity of Rec across Cas9 paralogs. Ultimately, this work provides an avenue by which to modulate the structure, motion, and guide RNA interactions at the level of the Rec lobe of Cas9, setting the stage for future studies of Cas9 variants and their effect on its allosteric mechanism.

PubMed: 40387084
DOI: 10.7554/eLife.99275

Experimental method

X-RAY DIFFRACTION (1.49 Å)