Allosteric Motions of the CRISPR-Cas9 HNH Nuclease Probed by NMR and Molecular Dynamics

Abstract

CRISPR-Cas9 is a widely employed genome-editing tool with functionality reliant on the ability of the Cas9 endonuclease to introduce site-specific breaks in double-stranded DNA.In this system, an intriguing allosteric communication has been suggested to control its DNA cleavage activity through flexibility of the catalytic HNH domain. Here, solution NMR experiments and a novel Gaussian accelerated Molecular Dynamics (GaMD) simulation method are used to capture the structural and dynamic determinants of allosteric signaling within the HNH domain. We reveal the existence of a millisecond timescale dynamic pathway that spans HNH from the region interfacing the adjacent RuvC nuclease and propagates up to the DNA recognition lobe in the full-length CRISPR-Cas9. These findings reveal a potential route of signal transduction within the CRISPR-Cas9 HNH nuclease, advancing our understanding of the allosteric pathway of activation. Further, considering the role of allosteric signaling in the specificity of CRISPR-Cas9, this work poses the mechanistic basis for novel engineering efforts aimed at improving its genome editing capability.

Molecular Dynamics Reveals a DNA-Induced Dynamic Switch Triggering Activation of CRISPR-Cas12a

“…In light of these results, our outcomes motivate future investigations to characterize the functional role of REC and Nuc in CRISPR-Cas12a. It is also notable that our data suggest an unforeseen allosteric communication in CRISPR-Cas12a, which we have previously described in CRISPR-Cas9, 35 , 40 , 41 in agreement with experimental data. 6 , 25 This hypothesis grants now in-depth investigations, which we are currently pursuing building on our interests in the computational determination of protein allostery.…”

“…Finally, it is interesting to note that only in the presence of complete nucleic acids (i.e., in the FnCas12a′, Figure 4 c), highly correlated motions are observed across the entire protein. This has also been established through MD simulations of the DNA-bound Cas9, 19 , 35 , 40 indicating a mechanism of interdomain allosteric communication. 41 , 42 This novel hypothesis in CRISPR-Cas12a arising from our data now warrants further computational investigations of protein allostery that are currently being pursued in our laboratory.…”

“…The Allnér force field 53 has been employed for Mg 2+ ions, and the TIP3P model 54 has been employed for waters. These force field parameters and the simulation protocol have also been employed in our recent studies of CRISPR-Cas9, 40 , 55 , 56 corroborated by NMR experiments and quantum mechanical calculations, enabling a fair comparison. An integration time step of 2 fs has been employed.…”

Unravelling the Graded Millisecond Allosteric Activation Mechanism of Imidazole Glycerol Phosphate Synthase

“…(11) The transient nature of the ternary complex and the allosteric transition of enzymes undergoing turnover hampers the structural and dynamic characterization of allosteric mechanisms and the identification of functionally relevant states. (12)(13)(14)(15)(16)(17) It is therefore not surprising that the allosterically active state remains hidden for several enzymes. Allosteric regulation operating in the model Thermotoga maritima Imidazole Glycerol Phosphate Synthase (IGPS) has been investigated from structural and dynamical perspectives.…”

Mechanisms of γ-Secretase Activation and Substrate Processing

“…GaMD has successfully revealed physical pathways and mechanisms of protein folding and ligand binding, which are consistent with experiments and long-timescale conventional MD simulations. 27,[33][34] It has also been applied to characterize protein-protein, [35][36] protein-membrane, 37 and protein-nucleic acid [38][39] interactions. Therefore, GaMD was applied in this study for enhanced sampling of the γ -secretase complex, a well-known slow enzyme.…”