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bioRxiv · 10.1101/660613

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

East, K. W.·Newton, J. C.·Morzan, U. N.·Acharya, A.·Skeens, E.·Jogl, G.·Batista, V. S.·Palermo, G.·Lisi, G. P.
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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 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.\n\n\n\nO_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=117 SRC=\"FIGDIR/small/660613v4_ufig1.gif\" ALT=\"Figure 1\">\nView larger version (64K):\norg.highwire.dtl.DTLVardef@13429c5org.highwire.dtl.DTLVardef@1a2493borg.highwire.dtl.DTLVardef@175e120org.highwire.dtl.DTLVardef@f1852_HPS_FORMAT_FIGEXP M_FIG C_FIG

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East, K. W., Newton, J. C., Morzan, U. N., Acharya, A., Skeens, E., Jogl, G., Batista, V. S., Palermo, G., Lisi, G. P.. 2019-06-05. Allosteric Motions of the CRISPR-Cas9 HNH Nuclease Probed by NMR and Molecular Dynamics. https://doi.org/10.1101/660613

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