RT Journal Article
SR Electronic
T1 Conformational Changes Regulate Metal Coordination in the Catalytic Sites of Cas9
JF bioRxiv
FD Cold Spring Harbor Laboratory
SP 2022.09.10.507422
DO 10.1101/2022.09.10.507422
A1 Anuska Das
A1 Jay Rai
A1 Mitchell O. Roth
A1 Yuerong Shu
A1 Megan L. Medina
A1 MacKenzie R. Barakat
A1 Hong Li
YR 2022
UL http://biorxiv.org/content/early/2022/09/11/2022.09.10.507422.abstract
AB The control of CRISPR-Cas9 activities plays an essential role in its safe adoption for clinical and research applications. Previous studies have identified conformational dynamics of Cas9 as a key regulatory element for its enzymatic activity. The molecular basis for how conformations influence the catalytic processes at both nuclease domains, however, remains elusive. Here we present well-resolved cryo-EM structures of the active Acidothermus cellulolyticus Cas9 (AceCas9) complexes representing pre-cleavage (2.9 Å), two reaction intermediate (2.2 Å and 2.4 Å) and two post-cleavage (2.6 Å and 2.7 Å) states that reveal active site rearrangement during DNA binding and phosphodiester bond breakage by Cas9. Strikingly, large domain rearrangements in Cas9 triggered by the cognate DNA result in subtle changes in active sites that facilitate coordination of metal ions required for catalysis. The two reaction intermediate structures further reveal small oscillations in domain conformations, which alternates the reactive states of the two catalytic centers, thereby coupling cleavage of the two DNA strands. Consistent with the roles of conformations in organizing the active sites, adjustments of metal coordination ligands lead to altered metal specificity.Competing Interest StatementThe authors have declared no competing interest.