Fig. 4: Structure and mechanism of Crn4.
From: Structure and mechanism of the broad spectrum CRISPR-associated ring nuclease Crn4
a Dimeric structure of Crn4b, with individual monomers coloured grey and gold. Each monomer has an extended β-hairpin arm wrapping around the other. The conserved residues H17 and R103 are positioned on loops above a central cavity. b Dimeric structure of Crn4a, with individual monomers coloured pink and blue. The conserved residues H15 and R1023 are positioned on loops above a central cavity. c Superimposition of a monomer of apo Crn4b (gold) and apo Crn4a (pink). Crn4a has a longer β-hairpin compared to Crn4b. The conserved residues H17 and R103 (Crnb4b) and H15 and R112 (Crn4a) are shown. d Dimeric structure of Crn4a in complex with cA6, with monomers coloured mauve and green. cA6 bound to Crn4a is shown in stick representation, with carbons coloured yellow. e Top-down view of the Crn4a dimer in complex with cA6 (same colouring as d), highlighting how the extended β-hairpin wraps around the neighbouring monomer. f Active site of Crn4a in complex with cA6, with monomers coloured green and mauve. R112 is suitably positioned to participate in cA6 binding and catalysis. A15 (Crn4a mutant in ligand-bound structure) is shown in mauve and H15 from the apo Crn4a structure is shown in pink; there is space for the histidine to swing into place to play a role in catalysis. T13 sits behind H15. g Superimposition of a monomer of apo Crn4a (pink) and Crn4a bound to cA6 (mauve), illustrating the movement of the β-hairpin upon binding of the ligand. Residues H15 and R112 are shown. h HPLC analysis of cleavage products of cA4 incubated with wild type Crn4a and T13A, H15A and R112A variants. Mutagenesis of H15 and R112 abolished ring nuclease activity, while the T13A variant was still active.
