Fig. 6: A model showing the central role of the BH in coordinating the conformational activation of Cas12a.

Cas12a undergoes largescale conformational changes that makes the bilobed protein to take a closed conformation after binding to crRNA, followed by an open conformation to enable R-loop growth, and finally attaining a slightly-less open state after full R-loop formation to reach the pre-catalytic state. Our work illuminates the molecular mechanisms by which BH (bridge helix, pink) orchestrates these conformational changes. a In the binary complex and during the initial phase of DNA binding, part of the BH is shorter with a disordered loop. b After DNA binding progresses ∼9 bp of RNA-DNA hybrid formation, (Supplementary Fig. 25), the long RuvC-H1 (RuvC-helix 1, purple) of RuvC motif-II unwinds a few turns and the BH remains shorter with the loop region. c When the R-loop attains ∼16 bp of hybridization, the BH elongates by gaining a few helical turns with its concomitant bending towards the crRNA-DNA hybrid. These changes of the BH are correlated to the loop-to-helical transitioning of the RuvC-lid (medium purple) through direct interactions between amino acids in the region of the BH that has undergone the helical transition, enabling opening of the RuvC active site pocket. RuvC-H1, especially K981, acts as a pivot by changing the residue that it interacts within the RuvC-lid before and after the helical transition of the RuvC-lid. In FnoCas12a^KD2P, where BH transition is hindered, the protein attains the open conformation (second state in the top panels) and the interactions in the pre-catalytic state are absent, with the RuvC-lid exiting in a loop form. The R-loop is only partially formed and distorted in FnoCas12a^KD2P, which demonstrates the allosteric cooperation of BH and RuvC-H1 to open the RuvC active site pocket based on the number of base pairs in the RNA-DNA hybrid. In this figure, interactions for panels a and b are for FnoCas12a^KD2P (with K969P and D970P substitutions), while that in (c) is for FnoCas12a^WT20 (with K969 and D970). Figure was prepared using Microsoft PowerPoint.