Fig. 5: AceCas9 reaction scheme derived from the observed reactive states.
From: Coupled catalytic states and the role of metal coordination in Cas9
From left to right, the correctly placed HNH ___domain from the OPEN (state A) to the CLOSED conformation initiates catalysis that begins by nucleophilic attack, followed by cleavage intermediates and, ultimately, relaxation back to the OPEN conformation (states C1 and C2). The same colouring scheme for domains and nucleic acids as that in Figs. 1 and 2 is used for the cartoon. The use of wild-type enzyme under reaction-prone conditions prevented capture of the substrate–enzyme complex, although two intermediates (states B1 and B2), as shown in square brackets, were trapped immediately after nucleophilic attack. ‘B’ and ‘P’ in nucleotide structures indicate base and phosphate, respectively. The grey-coloured leaving nucleotide at the RuvC site reflects the fact that it is not captured in the structures, possibly due to the known 3′–5′ exonuclease activity of RuvC or its disorder. A slight but noticeable rotation of the HNH ___domain from that in the first intermediate B1 to that in the second intermediate B2 (conformation adjustment) relaxes the HNH and RuvC metal-coordination spheres towards the enzyme–product state. The green arrow indicates the conformation-dependent glutamate of the RuvC centre. Red dashed lines with marked distances highlight key changes in the reactive states. M, metal; MA, metal A; MB, metal B.
