Structural and functional investigation of GajB protein in Gabija 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.

The structure of AcrIE4-F7 reveals a common strategy for dual CRISPR inhibition by targeting PAM recognition sites.

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 αß 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.

Structural and mechanistic insights into the CRISPR inhibition of AcrIF7.

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 ß1ß2α1α2ß3 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.

Intrinsic disorder is essential for Cas9 inhibition of anti-CRISPR AcrIIA5.

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/ß 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.

Molecular organization of the type II-A CRISPR adaptation module and its interaction with Cas9 via Csn2.

Clustered regularly interspaced short palindromic repeats (CRISPRs) and CRISPR-associated (Cas) proteins provide microbial adaptive immunity against invading foreign nucleic acids. In type II-A CRISPR-Cas systems, the Cas1-Cas2 integrase complex and the subtype-specific Csn2 comprise the CRISPR adaptation module, which cooperates with the Cas9 nuclease effector for spacer selection. Here, we report the molecular organization of the Streptococcus pyogenes type II-A CRISPR adaptation module and its interaction with Cas9 via Csn2. We determined the crystal structure of S. pyogenes type II-A Cas2. Chromatographic and calorimetric analyses revealed the stoichiometry and topology of the type II-A adaptation module composed of Cas1, Cas2 and Csn2. We also demonstrated that Cas9 interacts with Csn2 in a direct and stoichiometric manner. Our results reveal a network of molecular interactions among type II-A Cas proteins and highlight the role of Csn2 in coordinating Cas components involved in the adaptation and interference stages of CRISPR-mediated immunity.

Crystal structure of an anti-CRISPR protein, AcrIIA1.

Clustered regularly interspaced short palindromic repeats (CRISPRs) and CRISPR-associated (Cas) proteins provide bacteria with RNA-based adaptive immunity against phage infection. To counteract this defense mechanism, phages evolved anti-CRISPR (Acr) proteins that inactivate the CRISPR-Cas systems. AcrIIA1, encoded by Listeria monocytogenes prophages, is the most prevalent among the Acr proteins targeting type II-A CRISPR-Cas systems and has been used as a marker to identify other Acr proteins. Here, we report the crystal structure of AcrIIA1 and its RNA-binding affinity. AcrIIA1 forms a dimer with a novel two helical-domain architecture. The N-terminal domain of AcrIIA1 exhibits a helix-turn-helix motif similar to transcriptional factors. When overexpressed in Escherichia coli, AcrIIA1 associates with RNAs, suggesting that AcrIIA1 functions via nucleic acid recognition. Taken together, the unique structural and functional features of AcrIIA1 suggest its distinct mode of Acr activity, expanding the diversity of the inhibitory mechanisms employed by Acr proteins.

CRISPR RNA and anti-CRISPR protein binding to the Xanthomonas albilineans Csy1-Csy2 heterodimer in the type I-F CRISPR-Cas system.

Clustered regularly interspaced short palindromic repeats (CRISPRs) and CRISPR-associated (Cas) proteins provide microbial adaptive immunity against bacteriophages. In type I-F CRISPR-Cas systems, multiple Cas proteins (Csy1-4) compose a surveillance complex (Csy complex) with CRISPR RNA (crRNA) for target recognition. Here, we report the biochemical characterization of the Csy1-Csy2 subcomplex from Xanthomonas albilineans, including the analysis of its interaction with crRNA and AcrF2, an anti-CRISPR (Acr) protein from a phage that infects Pseudomonas aeruginosa The X. albilineans Csy1 and Csy2 proteins (XaCsy1 and XaCsy2, respectively) formed a stable heterodimeric complex that specifically bound the 8-nucleotide (nt) 5'-handle of the crRNA. In contrast, the XaCsy1-XaCsy2 heterodimer exhibited reduced affinity for the 28-nt X. albilineans CRISPR repeat RNA containing the 5'-handle sequence. Chromatographic and calorimetric analyses revealed tight binding between the Acr protein from the P. aeruginosa phage and the heterodimeric subunit of the X. albilineans Csy complex, suggesting that AcrF2 recognizes conserved features of Csy1-Csy2 heterodimers. We found that neither XaCsy1 nor XaCsy2 alone forms a stable complex with AcrF2 and the 5'-handle RNA, indicating that XaCsy1-XaCsy2 heterodimerization is required for binding them. We also solved the crystal structure of AcrF2 to a resolution of 1.34 Å, enabling a more detailed structural analysis of the residues involved in the interactions with the Csy1-Csy2 heterodimer. Our results provide information about the order of events during the formation of the multisubunit crRNA-guided surveillance complex and suggest that the Acr protein inactivating type I-F CRISPR-Cas systems has broad specificity.

Enzyme intermediates captured "on the fly" by mix-and-inject serial crystallography.

BACKGROUND: Ever since the first atomic structure of an enzyme was solved, the discovery of the mechanism and dynamics of reactions catalyzed by biomolecules has been the key goal for the understanding of the molecular processes that drive life on earth. Despite a large number of successful methods for trapping reaction intermediates, the direct observation of an ongoing reaction has been possible only in rare and exceptional cases. RESULTS: Here, we demonstrate a general method for capturing enzyme catalysis "in action" by mix-and-inject serial crystallography (MISC). Specifically, we follow the catalytic reaction of the Mycobacterium tuberculosis ß-lactamase with the third-generation antibiotic ceftriaxone by time-resolved serial femtosecond crystallography. The results reveal, in near atomic detail, antibiotic cleavage and inactivation from 30 ms to 2 s. CONCLUSIONS: MISC is a versatile and generally applicable method to investigate reactions of biological macromolecules, some of which are of immense biological significance and might be, in addition, important targets for structure-based drug design. With megahertz X-ray pulse rates expected at the Linac Coherent Light Source II and the European X-ray free-electron laser, multiple, finely spaced time delays can be collected rapidly, allowing a comprehensive description of biomolecular reactions in terms of structure and kinetics from the same set of X-ray data.

An integrated approach for thermal stabilization of a mesophilic adenylate kinase.

Thermally stable proteins are desirable for research and industrial purposes, but redesigning proteins for higher thermal stability can be challenging. A number of different techniques have been used to improve the thermal stability of proteins, but the extents of stability enhancement were sometimes unpredictable and not significant. Here, we systematically tested the effects of multiple stabilization techniques including a bioinformatic method and structure-guided mutagenesis on a single protein, thereby providing an integrated approach to protein thermal stabilization. Using a mesophilic adenylate kinase (AK) as a model, we identified stabilizing mutations based on various stabilization techniques, and generated a series of AK variants by introducing mutations both individually and collectively. The redesigned proteins displayed a range of increased thermal stabilities, the most stable of which was comparable to a naturally evolved thermophilic homologue with more than a 25° increase in its thermal denaturation midpoint. We also solved crystal structures of three representative variants including the most stable variant, to confirm the structural basis for their increased stabilities. These results provide a unique opportunity for systematically analyzing the effectiveness and additivity of various stabilization mechanisms, and they represent a useful approach for improving protein stability by integrating the reduction of local structural entropy and the optimization of global noncovalent interactions such as hydrophobic contact and ion pairs.

Effectiveness and limitations of local structural entropy optimization in the thermal stabilization of mesophilic and thermophilic adenylate kinases.

Local structural entropy (LSE) is a descriptor for the extent of conformational heterogeneity in short protein sequences that is computed from structural information derived from the Protein Data Bank. Reducing the LSE of a protein sequence by introducing amino acid mutations can result in fewer conformational states and thus a more stable structure, indicating that LSE optimization can be used as a protein stabilization method. Here, we describe a series of LSE optimization experiments designed to stabilize mesophilic and thermophilic adenylate kinases (AKs) and report crystal structures of LSE-optimized AK variants. In the mesophilic AK, thermal stabilization by LSE reduction was effective but limited. Structural analyses of the LSE-optimized mesophilic AK variants revealed a strong correlation between LSE and the apolar buried surface area. Additional mutations designed to introduce noncovalent interactions between distant regions of the polypeptide resulted in further stabilization. Unexpectedly, optimizing the LSE of the thermophilic AK resulted in a decrease in thermal stability. This destabilization was reduced when charged residues were excluded from the possible substitutions during LSE optimization. These observations suggest that stabilization by LSE reduction may result from the optimization of local hydrophobic contacts. The limitations of this process are likely due to ignorance of other interactions that bridge distant regions in a given amino acid sequence. Our results illustrate the effectiveness and limitations of LSE optimization as a protein stabilization strategy and highlight the importance and complementarity of local conformational stability and global interactions in protein thermal stability.

Structural and functional characterization of Streptococcus pyogenes Cas2 protein under different pH conditions.

Clustered regularly interspaced short palindromic repeats (CRISPRs) and CRISPR-associated (Cas) proteins constitute an RNA-guided microbial defense system against invading foreign genetic materials. Cas2 is one of the core Cas proteins found universally in all the subtypes of CRISPR-Cas systems and is required for incorporating new spacers into CRISPR loci. Cas2 homologues from different CRISPR-Cas subtypes were characterized previously as metal-dependent nucleases with different substrate preferences, and it was proposed that a pH-dependent conformational change mediates metal binding and catalysis. Here, we report the crystal structures of Streptococcus pyogenes Cas2 at three different pHs (5.6, 6.5, and 7.5), as well as the results of its nuclease activity assay against double-stranded DNAs at varying pHs (6.0-9.0). Although S. pyogenes Cas2 exhibited strongly pH-dependent catalytic activity, there was no significant conformational difference among the three crystal structures. However, structural comparisons with other Cas2 homologues revealed structural variability and the flexible nature of its putative hinge regions, supporting the hypothesis that conformational switching is important for catalysis. Taken together, our results confirm that Cas2 proteins have pH-dependent nuclease activity against double-stranded DNAs, and provide indirect structural evidence for their conformational changes.

Conformational dynamics of adenylate kinase in crystals.

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.

Bioinformatic method for protein thermal stabilization by structural entropy optimization.

Engineering proteins for higher thermal stability is an important and difficult challenge. We describe a bioinformatic method incorporating sequence alignments to redesign proteins to be more stable through optimization of local structural entropy. Using this method, improved configurational entropy (ICE), we were able to design more stable variants of a mesophilic adenylate kinase with only the sequence information of one psychrophilic homologue. The redesigned proteins display considerable increases in their thermal stabilities while still retaining catalytic activity. ICE does not require a three-dimensional structure or a large number of homologous sequences, indicating a broad applicability of this method. Our results also highlight the importance of entropy in the stability of protein structures.

Structural Investigation of Self-Assembly and Target Binding of Anti-CRISPR AcrIIC2.

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.

Structural and functional evidence of bacterial antiphage protection by Thoeris defense system via NAD+ degradation.

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.

Structural and mutational analyses of psychrophilic and mesophilic adenylate kinases highlight the role of hydrophobic interactions in protein thermal stability.

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.

Structure and interactions of the first three RNA recognition motifs of splicing factor prp24.

The essential Saccharomyces cerevisiae pre-messenger RNA splicing protein 24 (Prp24) has four RNA recognition motifs (RRMs) and facilitates U6 RNA base-pairing with U4 RNA during spliceosome assembly. Prp24 is a component of the free U6 small nuclear ribonucleoprotein particle (snRNP) but not the U4/U6 bi-snRNP, and so is thought to be displaced from U6 by U4/U6 base-pairing. The interaction partners of each of the four RRMs of Prp24 and how these interactions direct U4/U6 pairing are not known. Here we report the crystal structure of the first three RRMs and the solution structure of the first two RRMs of Prp24. Strikingly, RRM 2 forms extensive inter-domain contacts with RRMs 1 and 3. These contacts occupy much of the canonical RNA-binding faces (beta-sheets) of RRMs 1 and 2, but leave the beta-sheet of RRM 3 exposed. Previously identified substitutions in Prp24 that suppress mutations in U4 and U6 spliceosomal RNAs cluster primarily in the beta-sheet of RRM 3, but also in a conserved loop of RRM 2. RNA binding assays and chemical shift mapping indicate that a large basic patch evident on the surface of RRMs 1 and 2 is part of a high affinity U6 RNA binding site. Our results suggest that Prp24 binds free U6 RNA primarily with RRMs 1 and 2, which may remodel the U6 secondary structure. The beta-sheet of RRM 3 then influences U4/U6 pairing through interaction with an unidentified ligand.

Solution structure and dynamics of anti-CRISPR AcrIIA4, the Cas9 inhibitor.

The bacterial CRISPR-Cas system provides adaptive immunity against invading phages. Cas9, an RNA-guided endonuclease, specifically cleaves target DNA substrates and constitutes a well-established platform for genome editing. Recently, anti-CRISPR (Acr) proteins that inhibit Cas9 have been discovered, promising a useful off-switch for Cas9 to avoid undesirable off-target effects. Here, we report the solution structure and dynamics of Listeria monocytogenes AcrIIA4 that inhibits Streptococcus pyogenes Cas9 (SpyCas9). AcrIIA4 forms a compact monomeric αßßßαα fold comprising three antiparallel ß strands flanked by three α-helices and a short 310-helix. AcrIIA4 exhibits distinct backbone dynamics in fast and slow timescales at loop regions that form interaction surfaces for SpyCas9. In particular, the ß1-ß2 loop that binds to the RuvC domain of SpyCas9 is highly mobile, and the ß1-ß2 and α2-α3 loops that bind to the RuvC and C-terminal domains of SpyCas9, respectively, undergoes conformational exchanges in microsecond-to-millisecond time scales. AcrIIA4 binds to apo-SpyCas9 with KD ~4.8 µM, which compares to KD ~0.6 nM for AcrIIA4 binding to sgRNA-bound SpyCas9. Since the binary complex between AcrIIA4 and SpyCas9 does not compete with the target DNA binding, it can effectively disable the Cas9 nuclease activity by forming a tight ternary complex in the presence of sgRNA.

Solution structure and dynamics of Xanthomonas albilineans Cas2 provide mechanistic insight on nuclease activity.

Cas2 protein in the CRISPR-Cas system functions as a scaffold for the acquisition of foreign DNA fragments, and as a nuclease against DNA and RNA substrates. Crystal structures of Cas2 have shown catalytically inactive conformational states that do not explain the mechanism of Cas2 nuclease activity. Here, we report that Xanthomonas albilineans Cas2 (XaCas2) assumes an inactive conformation in solution. Residual dipolar couplings and NMR relaxation, however, provide direct evidence on conformational dynamics at the predicted hinge region. Furthermore, XaCas2 transiently associates with metal ions for nuclease activity via highly mobile Asp8. Taken together, the dual function of Cas2 can be explained by a dynamic equilibrium of conformational states that serve as a scaffold or as a nuclease on demand.