CRISPR-Cas9 conformational activation as elucidated from enhanced molecular simulations

Abstract

CRISPR-Cas9 has become a facile genome editing technology, yet the structural and mechanistic features underlying its function are unclear. Here, we perform extensive molecular simulations in an enhanced sampling regime, using a Gaussian-accelerated molecular dynamics (GaMD) methodology, which probes displacements over hundreds of microseconds to milliseconds, to reveal the conformational dynamics of the endonuclease Cas9 during its activation toward catalysis. We disclose the conformational transition of Cas9 from its apo form to the RNA-bound form, suggesting a mechanism for RNA recruitment in which the domain relocations cause the formation of a positively charged cavity for nucleic acid binding. GaMD also reveals the conformation of a catalytically competent Cas9, which is prone for catalysis and whose experimental characterization is still limited. We show that, upon DNA binding, the conformational dynamics of the HNH domain triggers the formation of the active state, explaining how the HNH domain exerts a conformational control domain over DNA cleavage [Sternberg SH et al. (2015) Nature, 527, 110-113]. These results provide atomic-level information on the molecular mechanism of CRISPR-Cas9 that will inspire future experimental investigations aimed at fully clarifying the biophysics of this unique genome editing machinery and at developing new tools for nucleic acid manipulation based on CRISPR-Cas9.protein-nucleic acid interactions | gene regulation | RNA dynamics | enhanced sampling | free energy L ife sciences are undergoing a transformative phase due to an emerging genome editing technology based on the RNAprogrammable CRISPR-Cas9 (clustered regularly interspaced short palindromic repeats-CRISPR-associated protein 9) system (1-3). Although this facile genome editing technology is revolutionizing the fields of medicine, pharmaceutics, and even agriculture with the development of drought-resistant crops, the structural and mechanistic details underlying the CRISPR-Cas9 function remain unclear. The CRISPR-Cas9 function begins with the association of Cas9 with a guide RNA, composed of a CRISPR RNA (crRNA) and a transactivating CRISPR RNA (tracrRNA), which enables the recognition and cleavage of matching sequences in double-stranded DNA (3, 4). Upon site-specific recognition of a Protospacer Adjacent Motif (PAM) within the DNA, the latter binds Cas9 matching one strand with the RNA guide (the target DNA strand, t-DNA), whereas the other strand (nontarget DNA, nt-DNA) is displaced. Subsequently, two nuclease domains-HNH and RuvC-perform site-specific cleavages of the t-DNA and nt-DNA strands, respectively.Structural studies have revealed that Cas9 is a large multidomain protein, composed of a recognition lobe, which mediates the nucleic acid binding through three regions (RECI-III), and a nuclease lobe including the RuvC and HNH catalytic cores (Fig. 1) (5). An arginine rich helix (R-rich) bridges the two lobes, whereas the protein C-terminal (Cterm) and PAM-interacting (PI) domains take part in the DNA bindi...

Mechanistic Insights into Specific G Protein Interactions with Adenosine Receptors Revealed by Accelerated Molecular Simulations

“…Particularly, distances were calculated between the Cα atoms of residues Arg 3.50 and Glu 6. 30 , the center-of-mass (COM) distance between the receptor NPxxY motif and the last 5 residues of the Gα α5 helix, and the COM distance between the Gα (excluding residues in the αN helix) and Gβ (excluding residues 2-45 in the N-terminus) subunits. Root-mean-square fluctuations (RMSFs) were calculated for the protein residues and ligands, averaged over three independent GaMD simulations and color coded for schematic representation of each complex system (Figure 1).…”

“…b The R3.50-E6.30 distance is the distance between the Cα atoms of conserved residues Arg 3.50 and Glu 6. 30 in the receptors. c The α5 orientation angle is the angle between COMs of the receptor orthosteric pocket, the last 5 and first 5 residues of the Gα α5 helix, illustrated in Figure S8.…”

Enhanced proofreading governs CRISPR–Cas9 targeting accuracy

“…The HNH nuclease domain of SpCas9 undergoes a substantial conformational rearrangement upon target binding 12–15 , which activates the RuvC nuclease for concerted cleavage of both strands of the DNA 12,16 . It was previously shown that the HNH domain stably docks in its active state with an on-target substrate, but becomes loosely trapped in a catalytically-inactive conformational checkpoint when bound to mismatched targets 10,12 .…”

Gaussian Accelerated Molecular Dynamics: Theory, Implementation, and Applications

“…As demonstrated on the model systems, these implementations facilitate the applications of GaMD in enhanced sampling and free energy calculations of a wide range of biomolecular systems, such as proteins, lipid membrane, nucleic acids, virus particles and cellular complex structures. Ongoing applications of GaMD also include the clustered regularly interspaced short palindromic repeats-CRISPR associated protein 9 (CRISPR-Cas9) system 96 , HIV protease, protein kinases, the acyl carrier proteins, and so on.…”