Activation of Csm6 ribonuclease by cyclic nucleotide binding: in an emergency, twist to open

Stuart McQuarrie; Januka S. Athukoralage; Stephen A. McMahon; Shirley Graham; Katrin Ackermann; Bela E. Bode; Malcolm F. White; Tracey M. Gloster

doi:10.1093/nar/gkad739

Activation of Csm6 ribonuclease by cyclic nucleotide binding: in an emergency, twist to open

Stuart McQuarrie, Januka S. Athukoralage, Stephen A. McMahon, Shirley Graham, Katrin Ackermann, Bela E. Bode, Malcolm F. White, Tracey M. Gloster

Research output: Contribution to journal › Article › peer-review

Abstract

Type III CRISPR systems synthesize cyclic oligoadenylate (cOA) second messengers as part of a multi-faceted immune response against invading mobile genetic elements (MGEs). cOA activates non-specific CRISPR ancillary defence nucleases to create a hostile environment for MGE replication. Csm6 ribonucleases bind cOA using a CARF (CRISPR-associated Rossmann Fold) domain, resulting in activation of a fused HEPN (Higher Eukaryotes and Prokaryotes Nucleotide binding) ribonuclease domain. Csm6 enzymes are widely used in a new generation of diagnostic assays for the detection of specific nucleic acid species. However, the activation mechanism is not fully understood. Here we characterised the cyclic hexa-adenylate (cA6) activated Csm6' ribonuclease from the industrially important bacterium Streptococcus thermophilus. Crystal structures of Csm6' in the inactive and cA6 bound active states illuminate the conformational changes which trigger mRNA destruction. Upon binding of cA6, there is a close to 60° rotation between the CARF and HEPN domains, which causes the 'jaws' of the HEPN domain to open and reposition active site residues. Key to this transition is the 6H domain, a right-handed solenoid domain connecting the CARF and HEPN domains, which transmits the conformational changes for activation.

Original language	English
Pages (from-to)	10590-10605
Number of pages	16
Journal	Nucleic Acids Research
Volume	51
Issue number	19
Early online date	25 Sept 2023
DOIs	https://doi.org/10.1093/nar/gkad739
Publication status	Published - 27 Oct 2023

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McQuarrie_2023_NAR_Activation-of-Csm6_CC
Copyright © The Author(s) 2023. Published by Oxford University Press on behalf of Nucleic Acids Research. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (https://creativecommons.org/licenses/by/4.0/), which permits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited.
Final published version, 1.71 MBLicence: CC BY

1 Active
4 Finished

CBASS: ERC-2020-ADG. CBASS
White, M. (PI)
European Research Council
22/04/21 → 21/04/26
Project: Standard
Leverhulme Trust SRF 2022 - Tracey Glost: Expanding the structural biology toolkit: learning cryo-electron microscopy
Gloster, T. (PI)
The Royal Society
1/10/22 → 30/09/23
Project: Standard
Enabling Shaped Pulse Capability: Enabling Shaped Pulse Capability for Superior Biological Structural Determination Using EPR Spectroscopy.
Lovett, J. E. (PI), Bode, B. E. (CoI), Penedo, C. (CoI), Pitt, S. J. (CoI), Schwarz-Linek, U. (CoI), Smith, G. M. (CoI), Watson, A. J. B. (CoI) & White, M. (CoI)
BBSRC
1/10/20 → 30/09/21
Project: Standard

Activation of Csm6 ribonuclease by cyclic nucleotide binding: in an emergency, twist to open (EPR dataset)
Ackermann, K. (Creator) & Bode, B. E. (Creator), University of St Andrews, 5 Dec 2023
DOI: 10.17630/dd5a8ddf-4178-4602-8d90-407955431f3f
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File

Cite this

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title = "Activation of Csm6 ribonuclease by cyclic nucleotide binding: in an emergency, twist to open",

abstract = "Type III CRISPR systems synthesize cyclic oligoadenylate (cOA) second messengers as part of a multi-faceted immune response against invading mobile genetic elements (MGEs). cOA activates non-specific CRISPR ancillary defence nucleases to create a hostile environment for MGE replication. Csm6 ribonucleases bind cOA using a CARF (CRISPR-associated Rossmann Fold) domain, resulting in activation of a fused HEPN (Higher Eukaryotes and Prokaryotes Nucleotide binding) ribonuclease domain. Csm6 enzymes are widely used in a new generation of diagnostic assays for the detection of specific nucleic acid species. However, the activation mechanism is not fully understood. Here we characterised the cyclic hexa-adenylate (cA6) activated Csm6' ribonuclease from the industrially important bacterium Streptococcus thermophilus. Crystal structures of Csm6' in the inactive and cA6 bound active states illuminate the conformational changes which trigger mRNA destruction. Upon binding of cA6, there is a close to 60° rotation between the CARF and HEPN domains, which causes the 'jaws' of the HEPN domain to open and reposition active site residues. Key to this transition is the 6H domain, a right-handed solenoid domain connecting the CARF and HEPN domains, which transmits the conformational changes for activation.",

author = "Stuart McQuarrie and Athukoralage, {Januka S.} and McMahon, {Stephen A.} and Shirley Graham and Katrin Ackermann and Bode, {Bela E.} and White, {Malcolm F.} and Gloster, {Tracey M.}",

note = "Funding: Biotechnology and Biological Sciences Research Council [BB/T004789/1 to M.F.W. and T.M.G.]; European Research Council [101018608 to M.F.W.]; equipment was funded by BBSRC [BB/R013780/1 and BB/T017740/1]; T.M.G. is a recipient of a Royal Society Leverhulme Trust Senior Research Fellowship [SRF\R1\221056]. ",

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T1 - Activation of Csm6 ribonuclease by cyclic nucleotide binding

T2 - in an emergency, twist to open

AU - McQuarrie, Stuart

AU - Athukoralage, Januka S.

AU - McMahon, Stephen A.

AU - Graham, Shirley

AU - Ackermann, Katrin

AU - Bode, Bela E.

AU - White, Malcolm F.

AU - Gloster, Tracey M.

N1 - Funding: Biotechnology and Biological Sciences Research Council [BB/T004789/1 to M.F.W. and T.M.G.]; European Research Council [101018608 to M.F.W.]; equipment was funded by BBSRC [BB/R013780/1 and BB/T017740/1]; T.M.G. is a recipient of a Royal Society Leverhulme Trust Senior Research Fellowship [SRF\R1\221056].

PY - 2023/10/27

Y1 - 2023/10/27

N2 - Type III CRISPR systems synthesize cyclic oligoadenylate (cOA) second messengers as part of a multi-faceted immune response against invading mobile genetic elements (MGEs). cOA activates non-specific CRISPR ancillary defence nucleases to create a hostile environment for MGE replication. Csm6 ribonucleases bind cOA using a CARF (CRISPR-associated Rossmann Fold) domain, resulting in activation of a fused HEPN (Higher Eukaryotes and Prokaryotes Nucleotide binding) ribonuclease domain. Csm6 enzymes are widely used in a new generation of diagnostic assays for the detection of specific nucleic acid species. However, the activation mechanism is not fully understood. Here we characterised the cyclic hexa-adenylate (cA6) activated Csm6' ribonuclease from the industrially important bacterium Streptococcus thermophilus. Crystal structures of Csm6' in the inactive and cA6 bound active states illuminate the conformational changes which trigger mRNA destruction. Upon binding of cA6, there is a close to 60° rotation between the CARF and HEPN domains, which causes the 'jaws' of the HEPN domain to open and reposition active site residues. Key to this transition is the 6H domain, a right-handed solenoid domain connecting the CARF and HEPN domains, which transmits the conformational changes for activation.

AB - Type III CRISPR systems synthesize cyclic oligoadenylate (cOA) second messengers as part of a multi-faceted immune response against invading mobile genetic elements (MGEs). cOA activates non-specific CRISPR ancillary defence nucleases to create a hostile environment for MGE replication. Csm6 ribonucleases bind cOA using a CARF (CRISPR-associated Rossmann Fold) domain, resulting in activation of a fused HEPN (Higher Eukaryotes and Prokaryotes Nucleotide binding) ribonuclease domain. Csm6 enzymes are widely used in a new generation of diagnostic assays for the detection of specific nucleic acid species. However, the activation mechanism is not fully understood. Here we characterised the cyclic hexa-adenylate (cA6) activated Csm6' ribonuclease from the industrially important bacterium Streptococcus thermophilus. Crystal structures of Csm6' in the inactive and cA6 bound active states illuminate the conformational changes which trigger mRNA destruction. Upon binding of cA6, there is a close to 60° rotation between the CARF and HEPN domains, which causes the 'jaws' of the HEPN domain to open and reposition active site residues. Key to this transition is the 6H domain, a right-handed solenoid domain connecting the CARF and HEPN domains, which transmits the conformational changes for activation.

U2 - 10.1093/nar/gkad739

DO - 10.1093/nar/gkad739

M3 - Article

C2 - 37747760

AN - SCOPUS:85175355753

SN - 0305-1048

VL - 51

SP - 10590

EP - 10605

JO - Nucleic Acids Research

JF - Nucleic Acids Research

IS - 19

ER -

Activation of Csm6 ribonuclease by cyclic nucleotide binding: in an emergency, twist to open

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Projects

CBASS: ERC-2020-ADG. CBASS

Leverhulme Trust SRF 2022 - Tracey Glost: Expanding the structural biology toolkit: learning cryo-electron microscopy

Enabling Shaped Pulse Capability: Enabling Shaped Pulse Capability for Superior Biological Structural Determination Using EPR Spectroscopy.

Datasets

Activation of Csm6 ribonuclease by cyclic nucleotide binding: in an emergency, twist to open (EPR dataset)

Cite this