Allosteric activation of CRISPR-Cas12a requires the concerted movement of the bridge helix and helix 1 of the RuvC II domain.
Journal
Nucleic acids research
ISSN: 1362-4962
Titre abrégé: Nucleic Acids Res
Pays: England
ID NLM: 0411011
Informations de publication
Date de publication:
23 09 2022
23 09 2022
Historique:
accepted:
26
08
2022
revised:
19
08
2022
received:
23
02
2022
pubmed:
16
9
2022
medline:
28
9
2022
entrez:
15
9
2022
Statut:
ppublish
Résumé
Nucleases derived from the prokaryotic defense system CRISPR-Cas are frequently re-purposed for gene editing and molecular diagnostics. Hence, an in-depth understanding of the molecular mechanisms of these enzymes is of crucial importance. We focused on Cas12a from Francisella novicida (FnCas12a) and investigated the functional role of helix 1, a structural element that together with the bridge helix (BH) connects the recognition and the nuclease lobes of FnCas12a. Helix 1 is structurally connected to the lid domain that opens upon DNA target loading thereby activating the active site of FnCas12a. We probed the structural states of FnCas12a variants altered in helix 1 and/or the bridge helix using single-molecule FRET measurements and assayed the pre-crRNA processing, cis- and trans-DNA cleavage activity. We show that helix 1 and not the bridge helix is the predominant structural element that confers conformational stability of FnCas12a. Even small perturbations in helix 1 lead to a decrease in DNA cleavage activity while the structural integrity is not affected. Our data, therefore, implicate that the concerted remodeling of helix 1 and the bridge helix upon DNA binding is structurally linked to the opening of the lid and therefore involved in the allosteric activation of the active site.
Identifiants
pubmed: 36107767
pii: 6701592
doi: 10.1093/nar/gkac767
pmc: PMC9508855
doi:
Substances chimiques
Bacterial Proteins
0
CRISPR-Associated Proteins
0
RNA, Guide
0
DNA
9007-49-2
Endonucleases
EC 3.1.-
Types de publication
Journal Article
Research Support, Non-U.S. Gov't
Langues
eng
Sous-ensembles de citation
IM
Pagination
10153-10168Informations de copyright
© The Author(s) 2022. Published by Oxford University Press on behalf of Nucleic Acids Research.
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