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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.

SUBMITTER: McQuarrie S

PROVIDER: S-EPMC10702470 | biostudies-literature | 2023 Oct

REPOSITORIES: biostudies-literature

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

McQuarrie Stuart S Athukoralage Januka S JS McMahon Stephen A SA Graham Shirley S Ackermann Katrin K Bode Bela E BE White Malcolm F MF Gloster Tracey M TM

Nucleic acids research 20231001 19

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 ...[more]

PMID: 37747760

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Project description:Cyclic nucleotide-modulated ion channels play several essential physiological roles. They are involved in signal transduction in photoreceptors and olfactory sensory neurons as well as pacemaking activity in the heart and brain. Investigations of the molecular mechanism of their actions, including structural and electrophysiological characterization, are restricted by the availability of stable, purified protein obtained from accessible systems. Here, we establish that SthK, a cyclic nucleotide-gated (CNG) channel from Spirochaeta thermophila, is an excellent model for investigating the gating of eukaryotic CNG channels at the molecular level. The channel has high sequence similarity with its eukaryotic counterparts and was previously reported to be activated by cyclic nucleotides in patch-clamp experiments with Xenopus laevis oocytes. We optimized protein expression and purification to obtain large quantities of pure, homogeneous, and active recombinant SthK protein from Escherichia coli A negative-stain electron microscopy (EM) single-particle analysis indicated that this channel is a promising candidate for structural studies with cryo-EM. Using radioactivity and fluorescence flux assays, as well as single-channel recordings in lipid bilayers, we show that the protein is partially activated by micromolar concentrations of cyclic adenosine monophosphate (cAMP) and that channel activity is increased by depolarization. Unlike previous studies, we find that cyclic guanosine monophosphate (cGMP) is also able to activate SthK, but with much lower efficiency than cAMP. The distinct sensitivities to different ligands resemble eukaryotic CNG and hyperpolarization-activated and cyclic nucleotide-modulated channels. Using a fluorescence binding assay, we show that cGMP and cAMP bind to SthK with similar apparent affinities, suggesting that the large difference in channel activation by cAMP or cGMP is caused by the efficacy with which each ligand promotes the conformational changes toward the open state. We conclude that the functional characteristics of SthK reported here will permit future studies to analyze ligand gating and discrimination in CNG channels.

Dataset Information

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

Publications

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

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