QatABCD is a widespread anti-phage defense system in prokaryotes comprising four protein components. QatC, a signature component, is homologous to QueC, an enzyme involved in nucleobase modification during queuosine biosynthesis. QatA and QatD are predicted to function as an ATPase and a nuclease, respectively, while QatB lacks identifiable sequence motifs. Here, we report the structural and functional characterization of QatB and QatC. We determine the structure of QatC bound to the ATP analog AMPPNP and perform structure-guided functional assays. We further find that QatB and QatC form a stable heterodimer and solve the structure of the QatB-QatC complex. In addition to determining the structure of QatB, structural analysis suggests that it may serve as a substrate of QatC, implicating a potential regulatory mechanism. These findings provide structural and functional insights into QatB and QatC, laying a foundation for understanding the molecular mechanism of the QatABCD system in phage defense.

The CRISPR-Cas system employs RNA-guided endonucleases to protect bacteria and archaea from invading bacteriophages and plasmids. In response, bacteriophages have evolved anti-CRISPR proteins that inhibit diverse types of the CRISPR-Cas system. AcrIE5 was discovered from the mobile genetic elements of Pseudomonas aeruginosa, and potently inhibits the type I-E CRISPR-Cas system of P. aeruginosa. Here, we determined the solution structure of AcrIE5 using NMR spectroscopy, which adopts a novel αβ fold comprising three α-helices and two β-strands. AcrIE5 harbors a mobile loop between the β-strands that is conserved among homologs encoded by MGEs infecting Pseudomonas species, but truncated in homologs from MGEs of other bacteria. AlphaFold correctly reproduced the experimental structure of AcrIE5 and predicted its binding at a cleft formed by Cas8e, Cas7e, and Cas5e within the P. aeruginosa type I-E Cascade. The mobile loop and α-helices of AcrIE5 mediated key interactions with Cas8e at the PAM recognition site, as well as with adjacent Cas7e and Cas5e. AcrIE5 did not bind individual subunits of Cascade with high affinity, suggesting that it recognizes a composite interface of the functional Cascade assembly. Taken together, our findings suggest that AcrIE5 may compete with DNA binding to the PAM recognition site of type I-E Cascade, similar to AcrIE3 and AcrIE4, and also highlight a potential functional role of the conserved mobile loop in host-specific anti-CRISPR activity.

The CRISPR-Cas system is an adaptive immune system in prokaryotes that provides protection against bacteriophages. As a countermeasure, bacteriophages have evolved various anti-CRISPR proteins that neutralize CRISPR-Cas immunity. Here, we report the structural and functional investigation of AcrIE7, which inhibits the type I-E CRISPR-Cas system in Pseudomonas aeruginosa. We determined both crystal and solution structures of AcrIE7, which revealed a novel helical fold. In binding assays using various biochemical methods, AcrIE7 did not tightly interact with a single Cas component in the type I-E Cascade complex or the CRISPR adaptation machinery. In contrast, AlphaFold modeling with our experimentally determined AcrIE7 structure predicted that AcrIE7 interacts with Cas3 in the type I-E CRISPR-Cas system in P. aeruginosa. Our findings are consistent with a model where AcrIE7 inhibits Cas3 and also highlight the effectiveness and limitations of AlphaFold modeling.

The arms race between bacteria and bacteriophages has driven the evolution of both CRISPR-Cas systems and anti-CRISPR (Acr) proteins. AcrIE9, a type I-E Acr protein identified in Pseudomonas aeruginosa, inhibits Cascade-mediated DNA binding by interacting with the Cas7e subunit. However, its structural basis and precise inhibitory mechanism have remained unclear. Here, we report the crystal structure of AcrIE9 at 1.73 Å resolution, along with additional structural and biochemical analyses. AcrIE9 exists as both monomer and dimer in solution, while the crystal structure reveals a homodimeric assembly. Each protomer adopts a unique α/β architecture, and structural similarity searches indicate that AcrIE9 represents a previously uncharacterized protein fold. In vitro binding assays using individually purified type I-E Cas subunits from P. aeruginosa did not detect direct interaction with AcrIE9, including with Cas7e. These findings suggest that AcrIE9 may recognize a composite interface formed only within the intact Cascade complex, consistent with the AlphaFold3 prediction of multivalent interactions with Cas7e subunits. Taken together, this study provides the structural characterization of AcrIE9 and supports an inhibitory mechanism involving a multi-subunit binding surface on Cascade.

Anti-CRISPR (Acr) proteins are natural inhibitors of CRISPR-Cas systems, found in bacteriophages and other genetic elements. AcrIE3, identified in a Pseudomonas phage, inactivates the type I-E CRISPR-Cas system in Pseudomonas aeruginosa by engaging with the Cascade complex. However, its precise inhibition mechanism has remained elusive. In this study, we present a comprehensive structural and biochemical analysis of AcrIE3, providing mechanistic insight into its anti-CRISPR function. Our results reveal that AcrIE3 selectively binds to the Cas8e subunit of the Cascade complex. The crystal structure of AcrIE3 exhibits an all-helical fold with a negatively charged surface. Through extensive mutational analyses, we show that AcrIE3 interacts with the protospacer adjacent motif (PAM) recognition site in Cas8e through its negatively charged surface residues. These findings enhance our understanding of the structure and function of type I-E Acr proteins, suggesting PAM interaction sites as primary targets for divergent Acr inhibitors.

Adenylate kinase is a ubiquitous enzyme in living systems and undergoes dramatic conformational changes during its catalytic cycle. For these reasons, it is widely studied by genetic, biochemical, and biophysical methods, both experimental and theoretical. We have determined the basic crystal structures of three differently liganded states of adenylate kinase from Methanotorrus igneus, a hyperthermophilic organism whose adenylate kinase is a homotrimeric oligomer. The multiple copies of each protomer in the asymmetric unit of the crystal provide a unique opportunity to study the variation in the structure and were further analyzed using advanced crystallographic refinement methods and analysis tools to reveal conformational heterogeneity and, thus, implied dynamic behaviors in the catalytic cycle.

In bacteria and archaea, CRISPRs and Cas proteins constitute an adaptive immune system against invading foreign genetic materials, such as bacteriophages and plasmids. To counteract CRISPR-mediated immunity, bacteriophages encode anti-CRISPR (Acr) proteins that neutralize the host CRISPR–Cas systems. Several Acr proteins that act against type I-E CRISPR–Cas systems have been identified. Here, we describe the biochemical characterization of two type I-E Acr proteins, AcrIE2 and AcrIE4. We determined the crystal structure of AcrIE2 using single-wavelength anomalous diffraction and performed a structural comparison with the previously reported AcrIE2 structures solved by different techniques. Binding assays with type I-E Cas proteins were carried out for the target identification of AcrIE2. We also analyzed the interaction between AcrIE4 and its target Cas component using biochemical methods. Our findings corroborate and expand the knowledge on type I-E Acr proteins, illuminating diverse molecular mechanisms of inhibiting CRISPR-mediated prokaryotic anti-phage defense.

Bacteriophages (phages) are viruses that infect bacteria and archaea. To fend off invading phages, the hosts have evolved a variety of anti-phage defense mechanisms. Gabija is one of the most abundant prokaryotic antiviral systems and consists of two proteins, GajA and GajB. GajA has been characterized experimentally as a sequence-specific DNA endonuclease. Although GajB was previously predicted to be a UvrD-like helicase, its function is unclear. Here, we report the results of structural and functional analyses of GajB. The crystal structure of GajB revealed a UvrD-like domain architecture, including two RecA-like core and two accessory subdomains. However, local structural elements that are important for the helicase function of UvrD are not conserved in GajB. In functional assays, GajB did not unwind or bind various types of DNA substrates. We demonstrated that GajB interacts with GajA to form a heterooctameric Gabija complex, but GajB did not exhibit helicase activity when bound to GajA. These results advance our understanding of the molecular mechanism underlying Gabija anti-phage defense and highlight the role of GajB as a component of a multi-subunit antiviral complex in bacteria.

Bacteria and archaea use the CRISPR-Cas system to fend off invasions of bacteriophages and foreign plasmids. In response, bacteriophages encode anti-CRISPR (Acr) proteins that potently inhibit host Cas proteins to suppress CRISPR-mediated immunity. AcrIE4-F7, which was isolated from Pseudomonas citronellolis, is a fused form of AcrIE4 and AcrIF7 that inhibits both type I-E and type I-F CRISPR-Cas systems. Here, we determined the structure of AcrIE4-F7 and identified its Cas target proteins. The N-terminal AcrIE4 domain adopts a novel α-helical fold that targets the PAM interaction site of the type I-E Cas8e subunit. The C-terminal AcrIF7 domain exhibits an αβ fold like native AcrIF7, which disables target DNA recognition by the PAM interaction site in the type I-F Cas8f subunit. The two Acr domains are connected by a flexible linker that allows prompt docking onto their cognate Cas8 targets. Conserved negative charges in each Acr domain are required for interaction with their Cas8 targets. Our results illustrate a common mechanism by which AcrIE4-F7 inhibits divergent CRISPR-Cas types.

Anti-CRISPR (Acr) proteins are phage-borne inhibitors of the CRISPR-Cas immune system in archaea and bacteria. AcrIIC2 from prophages of Neisseria meningitidis disables the nuclease activity of type II-C Cas9, such that dimeric AcrIIC2 associates with the bridge helix (BH) region of Cas9 to compete with guide RNA loading. AcrIIC2 in solution readily assembles into oligomers of variable lengths, but the oligomeric states are not clearly understood. In this study, we investigated the dynamic assembly of AcrIIC2 oligomers, and identified key interactions underlying the self-association. We report that AcrIIC2 dimers associate into heterogeneous high-order oligomers with the equilibrium dissociation constant KD ∼8 μM. Oligomerization is driven by electrostatic interactions between charged residues, and rational mutagenesis produces a stable AcrIIC2 dimer with intact Cas9 binding. Remarkably, the BH peptide of Cas9 is unstructured in solution, and undergoes a coil-to-helix transition upon AcrIIC2 binding, revealing a unique folding-upon-binding mechanism for Acr recognition.

Abstract Clustered regularly interspaced short palindromic repeats (CRISPRs) and CRISPR-associated (Cas) proteins provide adaptive immunity to prokaryotes against invading phages and plasmids. As a countermeasure, phages have evolved anti-CRISPR (Acr) proteins that neutralize the CRISPR immunity. AcrIIA5, isolated from a virulent phage of Streptococcus thermophilus, strongly inhibits diverse Cas9 homologs, but the molecular mechanism underlying the Cas9 inhibition remains unknown. Here, we report the solution structure of AcrIIA5, which features a novel α/β fold connected to an N-terminal intrinsically disordered region (IDR). Remarkably, truncation of the N-terminal IDR abrogates the inhibitory activity against Cas9, revealing that the IDR is essential for Cas9 inhibition by AcrIIA5. Progressive truncations and mutations of the IDR illustrate that the disordered region not only modulates the association between AcrIIA5 and Cas9–sgRNA, but also alters the catalytic efficiency of the inhibitory complex. The length of IDR is critical for the Cas9–sgRNA recognition by AcrIIA5, whereas the charge content of IDR dictates the inhibitory activity. Conformational plasticity of IDR may be linked to the broad-spectrum inhibition of Cas9 homologs by AcrIIA5. Identification of the IDR as the main determinant for Cas9 inhibition expands the inventory of phage anti-CRISPR mechanisms.

The CRISPR-Cas system provides an adaptive immunity for bacteria and archaea against invading phages or foreign plasmids. In the type II CRISPR-Cas system, a single effector protein Cas9 and a guide RNA form an RNA-guided endonuclease complex that can degrade DNA targets of foreign origin. To avoid the Cas9-mediated destruction, phages evolved anti-CRISPR (Acr) proteins that neutralize the host bacterial immunity by inactivating the CRISPR-Cas system. Here we report the backbone H-1, N-15 , and C-13 resonance assignments of AcrIIA5 that inhibits the endonuclease activity of type II-A Streptococcus thermophilus Cas9 and also Streptococcus pyogenesis Cas9 using triple resonance nuclear magnetic resonance spectroscopy. The backbone chemical shifts of AcrIIA5 predict a disordered region at the N-terminus, followed by an alpha beta beta beta beta alpha beta beta beta fold.

CRISPRs and Cas proteins form a microbial adaptive immune system against phage infection. To counteract this defense mechanism, phages evolved anti-CRISPR (Acr) proteins that inhibit the CRISPR-Cas systems. Here, we report the crystal structure of AcrIF2, an Acr protein inactivating the type I-F CRISPR-Cas system in Pseudomonas aeruginosa. In the type I-F systems, multiple Cas proteins (Csy1-4) constitute a surveillance complex with a guide RNA for target recognition. We demonstrated that AcrIF2 binds strongly to Csy1-Csy2 subcomplexes from two Xanthomonas species. In the crystal structure of AcrIF2, the arrangement of Asp and Glu side chains resembles the negative charge distribution of the dsDNA backbone, confirming that AcrIF2 is a dsDNA mimic blocking target DNA recognition by the CRISPR surveillance complex. Our results corroborate the inhibitory mechanism employed by AcrIF2 and suggest its broad specificity against diverse type I-F CRISPR-Cas systems.

The intense arms race between bacteria and phages has led to the development of diverse antiphage defense systems in bacteria. Unlike well-known restriction-modification and CRISPR-Cas systems, recently discovered systems are poorly characterized. One such system is the Thoeris defense system, which consists of two genes, thsA and thsB. Here, we report structural and functional analyses of ThsA and ThsB. ThsA exhibits robust NAD+ cleavage activity and a two-domain architecture containing sirtuin-like and SLOG-like domains. Mutation analysis suggests that NAD+ cleavage is linked to the antiphage function of Thoeris. ThsB exhibits a structural resemblance to TIR domain proteins such as nucleotide hydrolases and Toll-like receptors, but no enzymatic activity is detected in our in vitro assays. These results further our understanding of the molecular mechanism underlying the Thoeris defense system, highlighting a unique strategy for bacterial antiphage resistance via NAD+ degradation.

Abstract The CRISPR–Cas system provides adaptive immunity for bacteria and archaea to combat invading phages and plasmids. Phages evolved anti-CRISPR (Acr) proteins to neutralize the host CRISPR–Cas immune system as a counter-defense mechanism. AcrIF7 in Pseudomonas aeruginosa prophages strongly inhibits the type I-F CRISPR–Cas system. Here, we determined the solution structure of AcrIF7 and identified its target, Cas8f of the Csy complex. AcrIF7 adopts a novel β1β2α1α2β3 fold and interacts with the target DNA binding site of Cas8f. Notably, AcrIF7 competes with AcrIF2 for the same binding interface on Cas8f without common structural motifs. AcrIF7 binding to Cas8f is driven mainly by electrostatic interactions that require position-specific surface charges. Our findings suggest that Acrs of divergent origin may have acquired specificity to a common target through convergent evolution of their surface charge configurations.

Protein thermal stability is an important field since thermally stable proteins are desirable in many academic and industrial settings. Information on protein thermal stabilization can be obtained by comparing homologous proteins from organisms living at distinct temperatures. Here, we report structural and mutational analyses of adenylate kinases (AKs) from psychrophilic Bacillus globisporus (AKp) and mesophilic Bacillus subtilis (AKm). Sequence and structural comparison showed suboptimal hydrophobic packing around Thr26 in the CORE domain of AKp, which was replaced with an Ile residue in AKm. Mutations that improved hydrophobicity of the Thr residue increased the thermal stability of the psychrophilic AKp, and the largest stabilization was observed for a Thr-to-Ile substitution. Furthermore, a reverse Ile-to-Thr mutation in the mesophilic AKm significantly decreased thermal stability. We determined the crystal structures of mutant AKs to confirm the impact of the residue substitutions on the overall stability. Taken together, our results provide a structural basis for the stability difference between psychrophilic and mesophilic AK homologues and highlight the role of hydrophobic interactions in protein thermal stability.

CRISPRs and Cas proteins constitute an RNA-guided microbial immune system against invading nucleic acids. Cas1 is a universal Cas protein found in all three types of CRISPR-Cas systems, and its role is implicated in new spacer acquisition during CRISPR-mediated adaptive immunity. Here, we report the crystal structure of Streptococcus pyogenes Cas1 (SpCas1) in a type II CRISPR-Cas system and characterize its interaction with S. pyogenes Csn2 (SpCsn2). The SpCas1 structure reveals a unique conformational state distinct from type I Cas1 structures, resulting in a more extensive dimerization interface, a more globular overall structure, and a disruption of potential metal-binding sites for catalysis. We demonstrate that SpCas1 directly interacts with SpCsn2, and identify the binding interface and key residues for Cas complex formation. These results provide structural information for a type II Cas1 protein, and lay a foundation for studying multiprotein Cas complexes functioning in type II CRISPR-Cas systems.