RESUMEN
Bacteria have evolved sophisticated adaptive immune systems, called CRISPR-Cas, that provide sequence-specific protection against phage infection. In turn, phages have evolved a broad spectrum of anti-CRISPRs that suppress these immune systems. Here we report structures of anti-CRISPR protein IF9 (AcrIF9) in complex with the type I-F CRISPR RNA-guided surveillance complex (Csy). In addition to sterically blocking the hybridization of complementary dsDNA to the CRISPR RNA, our results show that AcrIF9 binding also promotes non-sequence-specific engagement with dsDNA, potentially sequestering the complex from target DNA. These findings highlight the versatility of anti-CRISPR mechanisms utilized by phages to suppress CRISPR-mediated immune systems.
Asunto(s)
Bacterias/metabolismo , Bacteriófagos/metabolismo , Sistemas CRISPR-Cas , ADN/metabolismo , ARN Guía de Kinetoplastida/metabolismo , Proteínas Virales/metabolismo , Secuencia de Aminoácidos , Bacterias/genética , Bacterias/virología , Proteínas Bacterianas/química , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Bacteriófagos/genética , Microscopía por Crioelectrón , ADN/química , ADN/genética , Modelos Moleculares , Complejos Multiproteicos/química , Complejos Multiproteicos/metabolismo , Complejos Multiproteicos/ultraestructura , Conformación de Ácido Nucleico , Unión Proteica , Conformación Proteica , Proteus penneri/genética , Proteus penneri/metabolismo , Proteus penneri/virología , ARN Guía de Kinetoplastida/química , ARN Guía de Kinetoplastida/genética , Homología de Secuencia de Aminoácido , Proteínas Virales/química , Proteínas Virales/genéticaRESUMEN
Bacteria and archaea have evolved sophisticated adaptive immune systems that rely on CRISPR RNA (crRNA)-guided detection and nuclease-mediated elimination of invading nucleic acids. Here, we present the cryo-electron microscopy (cryo-EM) structure of the type I-F crRNA-guided surveillance complex (Csy complex) from Pseudomonas aeruginosa bound to a double-stranded DNA target. Comparison of this structure to previously determined structures of this complex reveals a â¼180-degree rotation of the C-terminal helical bundle on the "large" Cas8f subunit. We show that the double-stranded DNA (dsDNA)-induced conformational change in Cas8f exposes a Cas2/3 "nuclease recruitment helix" that is structurally homologous to a virally encoded anti-CRISPR protein (AcrIF3). Structural homology between Cas8f and AcrIF3 suggests that AcrIF3 is a mimic of the Cas8f nuclease recruitment helix.
Asunto(s)
Proteínas Bacterianas/metabolismo , Proteínas Asociadas a CRISPR/metabolismo , Sistemas CRISPR-Cas , Repeticiones Palindrómicas Cortas Agrupadas y Regularmente Espaciadas , ADN Bacteriano/metabolismo , Imitación Molecular , Pseudomonas aeruginosa/enzimología , ARN Bacteriano/metabolismo , ARN Guía de Kinetoplastida/metabolismo , Proteínas Virales/metabolismo , Proteínas Bacterianas/química , Proteínas Bacterianas/genética , Proteínas Bacterianas/inmunología , Proteínas Asociadas a CRISPR/química , Proteínas Asociadas a CRISPR/genética , Proteínas Asociadas a CRISPR/inmunología , Microscopía por Crioelectrón , ADN Bacteriano/química , ADN Bacteriano/genética , Modelos Moleculares , Conformación de Ácido Nucleico , Conformación Proteica , Pseudomonas aeruginosa/genética , Pseudomonas aeruginosa/inmunología , ARN Bacteriano/química , ARN Bacteriano/genética , ARN Guía de Kinetoplastida/química , ARN Guía de Kinetoplastida/genética , Relación Estructura-Actividad , Proteínas Virales/química , Proteínas Virales/genética , Proteínas Virales/inmunologíaRESUMEN
Bacteria and archaea rely on CRISPR (clustered regularly interspaced short palindromic repeats) RNA-guided adaptive immune systems for sequence specific elimination of foreign nucleic acids. In Escherichia coli, short CRISPR-derived RNAs (crRNAs) assemble with Cas (CRISPR-associated) proteins into a 405-kilodalton multisubunit surveillance complex called Cascade (CRISPR-associated complex for antiviral defense). Cascade binds foreign DNA complementary to the crRNA guide and recruits Cas3, a trans-acting nuclease-helicase required for target degradation. Structural models of Cascade have captured static snapshots of the complex in distinct conformational states, but conformational dynamics of the 11-subunit surveillance complex have not been measured. Here, we use hydrogen-deuterium exchange coupled to mass spectrometry (HDX-MS) to map conformational dynamics of Cascade onto the three-dimensional structure. New insights from structural dynamics are used to make functional predictions about the mechanisms of the R-loop coordination and Cas3 recruitment. We test these predictions in vivo and in vitro. Collectively, we show how mapping conformational dynamics onto static 3D-structures adds an additional dimension to the functional understanding of this biological machine.
Asunto(s)
Proteínas Asociadas a CRISPR/metabolismo , ADN Helicasas/metabolismo , ADN de Cadena Simple/metabolismo , Proteínas de Unión al ADN/metabolismo , ADN/metabolismo , Proteínas de Escherichia coli/metabolismo , Complejos Multiproteicos/metabolismo , ADN/química , ADN de Cadena Simple/química , Proteínas de Unión al ADN/genética , Deuterio , Escherichia coli/enzimología , Espectrometría de Masas , Complejos Multiproteicos/genética , Mutación , Conformación de Ácido Nucleico , Unión ProteicaRESUMEN
The type I-F CRISPR adaptive immune system in Pseudomonas aeruginosa (PA14) consists of two CRISPR loci and six CRISPR-associated (cas) genes. Type I-F systems rely on a CRISPR RNA (crRNA)-guided surveillance complex (Csy complex) to bind foreign DNA and recruit a trans-acting nuclease (i.e., Cas2/3) for target degradation. In most type I systems, Cas2 and Cas3 are separate proteins involved in adaptation and interference, respectively. However, in I-F systems, these proteins are fused into a single polypeptide. Here we use biochemical and structural methods to show that two molecules of Cas2/3 assemble with four molecules of Cas1 (Cas2/32:Cas14) into a four-lobed propeller-shaped structure, where the two Cas2 domains form a central hub (twofold axis of symmetry) flanked by two Cas1 lobes and two Cas3 lobes. We show that the Cas1 subunits repress Cas2/3 nuclease activity and that foreign DNA recognition by the Csy complex activates Cas2/3, resulting in bidirectional degradation of DNA targets. Collectively, this work provides a structure of the Cas1-2/3 complex and explains how Cas1 and the target-bound Csy complex play opposing roles in the regulation of Cas2/3 nuclease activity.
Asunto(s)
Proteínas Bacterianas/metabolismo , Sistemas CRISPR-Cas/fisiología , Desoxirribonucleasas/metabolismo , Complejos Multienzimáticos/metabolismo , Pseudomonas aeruginosa/enzimología , Proteínas Bacterianas/genética , Desoxirribonucleasas/genética , Complejos Multienzimáticos/genética , Pseudomonas aeruginosa/genéticaRESUMEN
Genetic conflict between viruses and their hosts drives evolution and genetic innovation. Prokaryotes evolved CRISPR-mediated adaptive immune systems for protection from viral infection, and viruses have evolved diverse anti-CRISPR (Acr) proteins that subvert these immune systems. The adaptive immune system in Pseudomonas aeruginosa (type I-F) relies on a 350 kDa CRISPR RNA (crRNA)-guided surveillance complex (Csy complex) to bind foreign DNA and recruit a trans-acting nuclease for target degradation. Here, we report the cryo-electron microscopy (cryo-EM) structure of the Csy complex bound to two different Acr proteins, AcrF1 and AcrF2, at an average resolution of 3.4 Å. The structure explains the molecular mechanism for immune system suppression, and structure-guided mutations show that the Acr proteins bind to residues essential for crRNA-mediated detection of DNA. Collectively, these data provide a snapshot of an ongoing molecular arms race between viral suppressors and the immune system they target.
Asunto(s)
Bacteriófagos/química , Proteínas Asociadas a CRISPR/química , Repeticiones Palindrómicas Cortas Agrupadas y Regularmente Espaciadas , Pseudomonas aeruginosa/inmunología , Pseudomonas aeruginosa/virología , ARN Bacteriano/química , Proteínas Virales/química , Bacteriófagos/clasificación , Bacteriófagos/genética , Microscopía por Crioelectrón , Cristalografía por Rayos X , Vigilancia Inmunológica , Modelos Moleculares , Pseudomonas aeruginosa/genética , ARN Bacteriano/metabolismo , ARN Bacteriano/ultraestructura , Proteínas Virales/ultraestructuraRESUMEN
In bacteria and archaea, short fragments of foreign DNA are integrated into Clustered Regularly Interspaced Short Palindromic Repeat (CRISPR) loci, providing a molecular memory of previous encounters with foreign genetic elements. In Escherichia coli, short CRISPR-derived RNAs are incorporated into a multi-subunit surveillance complex called Cascade (CRISPR-associated complex for antiviral defense). Recent structures of Cascade capture snapshots of this seahorse-shaped RNA-guided surveillance complex before and after binding to a DNA target. Here we determine a 3.2 Å x-ray crystal structure of Cascade in a new crystal form that provides insight into the mechanism of double-stranded DNA binding. Molecular dynamic simulations performed using available structures reveal functional roles for residues in the tail, backbone and belly subunits of Cascade that are critical for binding double-stranded DNA. Structural comparisons are used to make functional predictions and these predictions are tested in vivo and in vitro. Collectively, the results in this study reveal underlying mechanisms involved in target-induced conformational changes and highlight residues important in DNA binding and protospacer adjacent motif recognition.
Asunto(s)
Proteínas Asociadas a CRISPR/química , Repeticiones Palindrómicas Cortas Agrupadas y Regularmente Espaciadas , ADN/química , Proteínas de Escherichia coli/química , Escherichia coli/genética , Arginina/química , Proteínas Asociadas a CRISPR/metabolismo , ADN/metabolismo , Proteínas de Escherichia coli/metabolismo , Lisina/química , Modelos Moleculares , Conformación de Ácido Nucleico , Motivos de Nucleótidos , Unión Proteica , Conformación Proteica , ARN Bacteriano/metabolismoRESUMEN
Clustered regularly interspaced short palindromic repeats (CRISPRs) are essential components of RNA-guided adaptive immune systems that protect bacteria and archaea from viruses and plasmids. In Escherichia coli, short CRISPR-derived RNAs (crRNAs) assemble into a 405-kilodalton multisubunit surveillance complex called Cascade (CRISPR-associated complex for antiviral defense). Here we present the 3.24 angstrom resolution x-ray crystal structure of Cascade. Eleven proteins and a 61-nucleotide crRNA assemble into a seahorse-shaped architecture that binds double-stranded DNA targets complementary to the crRNA-guide sequence. Conserved sequences on the 3' and 5' ends of the crRNA are anchored by proteins at opposite ends of the complex, whereas the guide sequence is displayed along a helical assembly of six interwoven subunits that present five-nucleotide segments of the crRNA in pseudo-A-form configuration. The structure of Cascade suggests a mechanism for assembly and provides insights into the mechanisms of target recognition.