Figure 4. AcrVA4 locks the Cas12a bridge-helix to prevent DNA binding.

(A) AcrVA4 (surface) is shown braced against the bridge-helix (BH, green, cartoon) near the RuvC (teal, cartoon). The conformation of the LbCas12a bridge-helix when bound to DNA is shown semi-transparent with a green arrow denoting the direction of helix motion upon DNA binding. (B) Detailed atomistic view of AcrVA4 (red) recognition of the Cas12a bridge-helix (green). (C) LbCas12a dsDNA cis-cleavage over time measured under single-turnover conditions in the presence or absence of AcrVA4 containing alanine substitutions (mean ∓ s.d., n = 3 independent measurements). Two-phase exponential decay experimental fits are shown as solid or dashed lines. (D) Size-exclusion chromatography coupled di-angle light scattering (SEC-DALS) trace for AcrVA4 W178A in the presence of LbCas12a-crRNA. The absorbance at 280 nm (blue) and 260 nm (gray) are shown (left axis) with the linear region for the mass estimate corresponding to the relevant peaks (black lines, right axis).

Figure 4—figure supplement 1. Superposition of LbCas12a-crRNA (PDB code: 5ID6) on our State I LbCas12a-crRNA-AcrVA4 complex.

(A) Both the LbCas12a-crRNA complex (5ID6) and our State I structure are shown as cartoons with the domains colored as in Figure 1. (B) A subtle distortion of the loop between the LbCas12a bridge-helix (cartoon, green) and RuvC (cartoon, teal) is observed in the presence of AcrVA4 (surface, red). R887 and Q888 adopt different conformations between the LbCas12a-crRNA complex (sticks, white) and the LbCas12a-crRNA-AcrVA4 complex (sticks, green).

Figure 4—figure supplement 2. Size exclusion chromatography coupled di-angle light scattering (SEC-DALS) for AcrVA4 and LbCas12a-crRNA.

Size exclusion chromatography coupled light scattering traces for (A) AcrVA4, (B) AcrVA4 (W178A), (C) LbCas12a-crRNA complex, (D) LbCas12a-crRNA-AcrVA4 complex, and (E) LbCas12a-crRNA-AcrVA4 (W178A) complex. For each plot, the absorbance at 260 nm (gray) and 280 nm (blue) are shown (left axis) with the linear region for the mass estimate (black lines) corresponding to the relevant peaks (right axis). The theoretical and calculated molecular weights are annotated on each graph.

Figure 4—video 1. LbCas12a undergoes a large conformational change from the crRNA-bound state to a DNA-bound state.

Download video file ^{(1.3MB, mp4)}

DOI: 10.7554/eLife.49110.014

Structural morph illustrating the conformational change between the LbCas12a-crRNA complex (PDB code: 5ID6) and the LbCas12a-crRNA-DNA complex (PDB code: 5XUS). Protein domains are colored as in Figure 1. The crRNA phosphate backbone is shown in orange with nucleobases in the cartoon ring representation.

Figure 4—video 2. The LbCas12a bridge-helix undergoes a large conformational change upon DNA binding.

Download video file ^{(1.1MB, mp4)}

DOI: 10.7554/eLife.49110.015

Structural morph illustrating the conformational change between the LbCas12a-crRNA complex (PDB code: 5ID6) and the LbCas12a-crRNA-DNA complex (PDB code: 5XUS). Protein domains are colored as in Figure 1. The bases of the crRNA and the target DNA strand are shown in orange and white, respectively. Bridge-helix Arg 887 is shown with a stick representation.

Figure 4—video 3. AcrVA4 binding prevents LbCas12a bridge-helix conformational dynamics upon DNA binding.

Download video file ^{(1.2MB, mp4)}

DOI: 10.7554/eLife.49110.016

Structural morph illustrating the conformational change between the LbCas12a-crRNA-AcrVA4 complex (PDB code: 6P7M) and the LbCas12a-crRNA-DNA complex (PDB code: 5XUS). Protein domains are colored as in Figure 1. Protein domains are colored as in Figure 1. The phosphate backbone of the crRNA and the target DNA strand are shown in orange and white, respectively. Bridge-helix Arg 887 is shown with a stick representation. AcrVA4 is shown as a semi-transparent surface.