Cat1 forms filament networks to degrade NAD+ during the type III CRISPR-Cas antiviral response.
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| Title: | Cat1 forms filament networks to degrade NAD+ during the type III CRISPR-Cas antiviral response. |
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| Authors: | Baca, Christian F., Majumder, Puja, Hickling, James H., Patel, Dinshaw J., Marraffini, Luciano A. |
| Source: | Science. 6/12/2025, Vol. 388 Issue 6752, p1-14. 14p. |
| Subjects: | CRISPRs, Oligoadenylates, Virus diseases, Prokaryotes, Staphylococcus |
| Abstract: | Type III CRISPR-Cas systems defend against viral infection in prokaryotes by using an RNA-guided complex that recognizes foreign transcripts and synthesizes cyclic oligoadenylate (cOA) messengers to activate CRISPR-associated Rossmann-fold (CARF) immune effectors. In this study, we investigated a protein containing a CARF domain–fused Toll/interleukin-1 receptor (TIR) domain, Cat1. We found that Cat1 provides immunity by cleaving and depleting oxidized nicotinamide adenine dinucleotide (NAD+) molecules from the infected host, inducing a growth arrest that prevents viral propagation. Cat1 forms dimers that stack upon each other to generate long filaments that are maintained by bound cOA ligands, with stacked TIR domains forming the NAD+ cleavage catalytic sites. Furthermore, Cat1 filaments assemble into distinct trigonal and pentagonal networks that enhance NAD+ degradation. Cat1 presents an unprecedented chemistry and higher-order protein assembly for the CRISPR-Cas response. Editor's summary: CRISPR-Cas systems defend bacteria and archaea against viral (phage) infection using RNA-guided complexes. In many cases, such as in CRISPR-Cas9, the complex directly destroys the phage genome to provide immunity. In the case of CRISPR-Cas10, the RNA-guided complex synthesizes cyclic nucleotide second messengers that activate the downstream effectors responsible for the interruption of the phage infectious cycle. Baca et al. describe a CRISPR-associated TIR protein effector, Cat1, which, upon binding of the cyclic nucleotide ligand, forms long filaments that assemble into large networks formed by trigonal and pentagonal filament bundles. The TIR domains within these filaments cleave nicotinamide adenine dinucleotide, depleting this essential metabolite from the infected host to prevent viral propagation. —Di Jiang INTRODUCTION: CRISPR-Cas systems defend bacteria and archaea against viral (phage) infection using RNA-guided complexes that can be classified into different types depending on their molecular mechanism of immunity. In many cases, such as in type II CRISPR-Cas systems, the Cas9 ribonucleoprotein complex directly destroys the phage genome to provide immunity. For type III CRISPR-Cas systems, the Cas10 complex recognizes phage transcripts produced during infection that are complementary to the RNA guide. Target recognition triggers the cyclase activity of the Cas10 complex, which synthesizes cyclic oligoadenylates (cOAs) that act as second messengers to stimulate downstream CRISPR-associated Rossmann-fold (CARF) effectors. These effectors are composed of (i) a CARF domain that dimerizes to generate a cOA binding pocket and (ii) additional domains with enzymatic activities that, upon ligand binding, alter the host to interrupt the phage infectious cycle. Type III CRISPR-Cas systems are associated with an immense repertoire of CARF effectors that display diverse activities such as DNA and/or RNA cleavage, membrane depolarization, and adenosine triphosphate (ATP) deamination. Many other CARF effectors, however, remain uncharacterized. RATIONALE: We studied an effector containing a CARF domain fused to a Toll/interleukin-1 receptor (TIR) domain, Cat1. Previous work showed that TIR domains in bacteria and plants cleave oxidized nicotinamide adenine dinucleotide (NAD+) molecules. We expressed Cat1 in staphylococci carrying a type III-A CRISPR-Cas system and evaluated the effects of its activation and antiphage properties in vivo. We also purified the protein to determine cOA binding, enzymatic properties, and cryo–electron microscopy structure. RESULTS: Upon activation of cOA production, NAD+ is substantially depleted in staphylococci expressing Cat1, leading to a stop in cell division and an arrest of the growth of the bacterial culture. Cat1's effects on infected hosts interrupt the viral lytic cycle and prevent phage propagation, thus enabling the continuous growth of uninfected cells. In vitro, purified Cat1 binds cyclic tetra-adenylate (cA4), which activates the enzymatic cleavage of NAD+ into nicotinamide (NAM) and adenosine diphosphate ribose (ADPR). Alanine substitutions of residues lining both the cA4 binding pocket and the NAD+ cleavage site abrogate Cat1-mediated growth arrest. Upon ligand binding, Cat1 dimers stack upon each other to generate long filaments that are maintained by the bound cA4. Stacked TIR domains, which are radially arranged in the filament, form the NAD+ cleavage catalytic sites. Furthermore, Cat1 filaments can associate through backbone interactions between the CARF domains of one filament and the TIR domains of the adjacent filament to assemble into large networks formed by trigonal and pentagonal filament bundles. Deletion of the residues involved in this interaction disrupted the formation of the filament network and dramatically reduced NAD+ cleavage. CONCLUSION: Other prokaryotic defense systems also trigger filamentation of TIR domains to degrade NAD+ and prevent phage propagation, for example the TIR-SAVED and TIR-STING effectors associated with cyclic oligonucleotide–based antiphage signaling systems (CBASS). Whereas these effectors share a filament architecture in which the cyclic nucleotide–binding domains (SAVED and STING) are arranged on one side of the filament and the TIR catalytic domains are displayed on the other side of the filament, in Cat1 the cyclic nucleotide–binding domains are at the center of the filament with the catalytic TIR domains radially arranged. In addition, Cat1-mediated NAD+ cleavage presents a novel chemistry (NAD+ cleavage) not previously associated with the CRISPR-Cas response. The structure of Cat1 is also distinctive. First, in contrast to other CARF effectors, the cA4 binding pocket is formed by not one but two CARF dimers in which the bottom CARF dimer forms the base of the pocket and the top CARF dimer caps the ligand. Therefore, cA4 binding facilitates the stacking of CARF dimers into a long filament. Second, filaments assemble into large networks formed by trigonal and pentagonal filament bundles that dramatically enhance NAD+ cleavage. How this network increases the rate of NAD+ degradation is currently not known. Our results reveal that the CRISPR-Cas response, once thought to act exclusively on nucleic acids, can be mediated by a diverse range of molecular mechanisms. Cat1 filament networks provide immunity against phage infection.: During the type III CRISPR-Cas response, recognition of a target phage transcript by the guide RNA of the Cas10 complex triggers the synthesis of cyclic tetra-adenylates (cA4) that bind to CARF effectors such as Cat1. Upon cA4 binding, Cat1 dimers stack on top of each other to form filamentous structures that further assemble into large networks formed by trigonal and pentagonal filament bundles. The TIR domains within these filaments degrade the essential metabolite NAD+ to generate an inhospitable host incapable of supporting phage replication. [ABSTRACT FROM AUTHOR] |
| Copyright of Science is the property of American Association for the Advancement of Science and its content may not be copied or emailed to multiple sites without the copyright holder's express written permission. Additionally, content may not be used with any artificial intelligence tools or machine learning technologies. However, users may print, download, or email articles for individual use. This abstract may be abridged. No warranty is given about the accuracy of the copy. Users should refer to the original published version of the material for the full abstract. (Copyright applies to all Abstracts.) | |
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| Items | – Name: Title Label: Title Group: Ti Data: Cat1 forms filament networks to degrade NAD<superscript>+</superscript> during the type III CRISPR-Cas antiviral response. – Name: Author Label: Authors Group: Au Data: <searchLink fieldCode="AR" term="%22Baca%2C+Christian+F%2E%22">Baca, Christian F.</searchLink><br /><searchLink fieldCode="AR" term="%22Majumder%2C+Puja%22">Majumder, Puja</searchLink><br /><searchLink fieldCode="AR" term="%22Hickling%2C+James+H%2E%22">Hickling, James H.</searchLink><br /><searchLink fieldCode="AR" term="%22Patel%2C+Dinshaw+J%2E%22">Patel, Dinshaw J.</searchLink><br /><searchLink fieldCode="AR" term="%22Marraffini%2C+Luciano+A%2E%22">Marraffini, Luciano A.</searchLink> – Name: TitleSource Label: Source Group: Src Data: <searchLink fieldCode="JN" term="%22Science%22">Science</searchLink>. 6/12/2025, Vol. 388 Issue 6752, p1-14. 14p. – Name: Subject Label: Subjects Group: Su Data: <searchLink fieldCode="DE" term="%22CRISPRs%22">CRISPRs</searchLink><br /><searchLink fieldCode="DE" term="%22Oligoadenylates%22">Oligoadenylates</searchLink><br /><searchLink fieldCode="DE" term="%22Virus+diseases%22">Virus diseases</searchLink><br /><searchLink fieldCode="DE" term="%22Prokaryotes%22">Prokaryotes</searchLink><br /><searchLink fieldCode="DE" term="%22Staphylococcus%22">Staphylococcus</searchLink> – Name: Abstract Label: Abstract Group: Ab Data: Type III CRISPR-Cas systems defend against viral infection in prokaryotes by using an RNA-guided complex that recognizes foreign transcripts and synthesizes cyclic oligoadenylate (cOA) messengers to activate CRISPR-associated Rossmann-fold (CARF) immune effectors. In this study, we investigated a protein containing a CARF domain–fused Toll/interleukin-1 receptor (TIR) domain, Cat1. We found that Cat1 provides immunity by cleaving and depleting oxidized nicotinamide adenine dinucleotide (NAD+) molecules from the infected host, inducing a growth arrest that prevents viral propagation. Cat1 forms dimers that stack upon each other to generate long filaments that are maintained by bound cOA ligands, with stacked TIR domains forming the NAD+ cleavage catalytic sites. Furthermore, Cat1 filaments assemble into distinct trigonal and pentagonal networks that enhance NAD+ degradation. Cat1 presents an unprecedented chemistry and higher-order protein assembly for the CRISPR-Cas response. Editor's summary: CRISPR-Cas systems defend bacteria and archaea against viral (phage) infection using RNA-guided complexes. In many cases, such as in CRISPR-Cas9, the complex directly destroys the phage genome to provide immunity. In the case of CRISPR-Cas10, the RNA-guided complex synthesizes cyclic nucleotide second messengers that activate the downstream effectors responsible for the interruption of the phage infectious cycle. Baca et al. describe a CRISPR-associated TIR protein effector, Cat1, which, upon binding of the cyclic nucleotide ligand, forms long filaments that assemble into large networks formed by trigonal and pentagonal filament bundles. The TIR domains within these filaments cleave nicotinamide adenine dinucleotide, depleting this essential metabolite from the infected host to prevent viral propagation. —Di Jiang INTRODUCTION: CRISPR-Cas systems defend bacteria and archaea against viral (phage) infection using RNA-guided complexes that can be classified into different types depending on their molecular mechanism of immunity. In many cases, such as in type II CRISPR-Cas systems, the Cas9 ribonucleoprotein complex directly destroys the phage genome to provide immunity. For type III CRISPR-Cas systems, the Cas10 complex recognizes phage transcripts produced during infection that are complementary to the RNA guide. Target recognition triggers the cyclase activity of the Cas10 complex, which synthesizes cyclic oligoadenylates (cOAs) that act as second messengers to stimulate downstream CRISPR-associated Rossmann-fold (CARF) effectors. These effectors are composed of (i) a CARF domain that dimerizes to generate a cOA binding pocket and (ii) additional domains with enzymatic activities that, upon ligand binding, alter the host to interrupt the phage infectious cycle. Type III CRISPR-Cas systems are associated with an immense repertoire of CARF effectors that display diverse activities such as DNA and/or RNA cleavage, membrane depolarization, and adenosine triphosphate (ATP) deamination. Many other CARF effectors, however, remain uncharacterized. RATIONALE: We studied an effector containing a CARF domain fused to a Toll/interleukin-1 receptor (TIR) domain, Cat1. Previous work showed that TIR domains in bacteria and plants cleave oxidized nicotinamide adenine dinucleotide (NAD+) molecules. We expressed Cat1 in staphylococci carrying a type III-A CRISPR-Cas system and evaluated the effects of its activation and antiphage properties in vivo. We also purified the protein to determine cOA binding, enzymatic properties, and cryo–electron microscopy structure. RESULTS: Upon activation of cOA production, NAD+ is substantially depleted in staphylococci expressing Cat1, leading to a stop in cell division and an arrest of the growth of the bacterial culture. Cat1's effects on infected hosts interrupt the viral lytic cycle and prevent phage propagation, thus enabling the continuous growth of uninfected cells. In vitro, purified Cat1 binds cyclic tetra-adenylate (cA4), which activates the enzymatic cleavage of NAD+ into nicotinamide (NAM) and adenosine diphosphate ribose (ADPR). Alanine substitutions of residues lining both the cA4 binding pocket and the NAD+ cleavage site abrogate Cat1-mediated growth arrest. Upon ligand binding, Cat1 dimers stack upon each other to generate long filaments that are maintained by the bound cA4. Stacked TIR domains, which are radially arranged in the filament, form the NAD+ cleavage catalytic sites. Furthermore, Cat1 filaments can associate through backbone interactions between the CARF domains of one filament and the TIR domains of the adjacent filament to assemble into large networks formed by trigonal and pentagonal filament bundles. Deletion of the residues involved in this interaction disrupted the formation of the filament network and dramatically reduced NAD+ cleavage. CONCLUSION: Other prokaryotic defense systems also trigger filamentation of TIR domains to degrade NAD+ and prevent phage propagation, for example the TIR-SAVED and TIR-STING effectors associated with cyclic oligonucleotide–based antiphage signaling systems (CBASS). Whereas these effectors share a filament architecture in which the cyclic nucleotide–binding domains (SAVED and STING) are arranged on one side of the filament and the TIR catalytic domains are displayed on the other side of the filament, in Cat1 the cyclic nucleotide–binding domains are at the center of the filament with the catalytic TIR domains radially arranged. In addition, Cat1-mediated NAD+ cleavage presents a novel chemistry (NAD+ cleavage) not previously associated with the CRISPR-Cas response. The structure of Cat1 is also distinctive. First, in contrast to other CARF effectors, the cA4 binding pocket is formed by not one but two CARF dimers in which the bottom CARF dimer forms the base of the pocket and the top CARF dimer caps the ligand. Therefore, cA4 binding facilitates the stacking of CARF dimers into a long filament. Second, filaments assemble into large networks formed by trigonal and pentagonal filament bundles that dramatically enhance NAD+ cleavage. How this network increases the rate of NAD+ degradation is currently not known. Our results reveal that the CRISPR-Cas response, once thought to act exclusively on nucleic acids, can be mediated by a diverse range of molecular mechanisms. Cat1 filament networks provide immunity against phage infection.: During the type III CRISPR-Cas response, recognition of a target phage transcript by the guide RNA of the Cas10 complex triggers the synthesis of cyclic tetra-adenylates (cA4) that bind to CARF effectors such as Cat1. Upon cA4 binding, Cat1 dimers stack on top of each other to form filamentous structures that further assemble into large networks formed by trigonal and pentagonal filament bundles. The TIR domains within these filaments degrade the essential metabolite NAD+ to generate an inhospitable host incapable of supporting phage replication. [ABSTRACT FROM AUTHOR] – Name: AbstractSuppliedCopyright Label: Group: Ab Data: <i>Copyright of Science is the property of American Association for the Advancement of Science and its content may not be copied or emailed to multiple sites without the copyright holder's express written permission. Additionally, content may not be used with any artificial intelligence tools or machine learning technologies. However, users may print, download, or email articles for individual use. This abstract may be abridged. No warranty is given about the accuracy of the copy. Users should refer to the original published version of the material for the full abstract.</i> (Copyright applies to all Abstracts.) |
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| RecordInfo | BibRecord: BibEntity: Identifiers: – Type: doi Value: 10.1126/science.adv9045 Languages: – Code: eng Text: English PhysicalDescription: Pagination: PageCount: 14 StartPage: 1 Subjects: – SubjectFull: CRISPRs Type: general – SubjectFull: Oligoadenylates Type: general – SubjectFull: Virus diseases Type: general – SubjectFull: Prokaryotes Type: general – SubjectFull: Staphylococcus Type: general Titles: – TitleFull: Cat1 forms filament networks to degrade NAD+ during the type III CRISPR-Cas antiviral response. Type: main BibRelationships: HasContributorRelationships: – PersonEntity: Name: NameFull: Baca, Christian F. – PersonEntity: Name: NameFull: Majumder, Puja – PersonEntity: Name: NameFull: Hickling, James H. – PersonEntity: Name: NameFull: Patel, Dinshaw J. – PersonEntity: Name: NameFull: Marraffini, Luciano A. IsPartOfRelationships: – BibEntity: Dates: – D: 12 M: 06 Text: 6/12/2025 Type: published Y: 2025 Identifiers: – Type: issn-print Value: 00368075 Numbering: – Type: volume Value: 388 – Type: issue Value: 6752 Titles: – TitleFull: Science Type: main |
| ResultId | 1 |
