CRISPR RNA binding drives structural ordering that primes Cas7-11 for target cleavage

Abstract

Type III-E CRISPR–Cas effectors, referred to as Cas7-11 or giant Repeat-Associated Mysterious Protein, are single proteins that cleave target RNAs (tgRNAs) without nonspecific collateral cleavage, opening new possibilities for RNA editing. Here, biochemical assays combined with amide hydrogen–deuterium exchange mass spectrometry (HDX–MS) experiments reveal the dynamics of apo Cas7-11. The HDX–MS results suggest a mechanism by which CRISPR RNA (crRNA) stabilizes the folded state of the protein and subsequent tgRNA binding remodels it to the active form. HDX–MS shows that the four Cas7 RNA recognition motif (RRM) folds are well-folded, but insertion sequences, including disordered catalytic loops and β-hairpins of the Cas7.2/Cas7.3 active sites, fold upon binding crRNA leading to stronger interactions at domain–domain interfaces, and folding of the Cas7.1 processing site. TgRNA binding causes conformational changes around the catalytic loops of Cas7.2 and Cas7.3. We show that Cas7-11 cannot independently process the CRISPR array and that binding of partially processed crRNA induces multiple states in Cas7-11 and reduces tgRNA cleavage. The insertion domain interacts most stably with mature crRNA. Finally, we show a crRNA-induced conformational change in one of the tetratricopeptide repeat fused with Cas/HEF1-associated signal transducer (TPR-CHAT) binding sites providing an explanation for why crRNA binding facilitates TPR-CHAT binding.