Type III-B CRISPR-Cas cascade of proteolytic cleavages

Abstract

The generation of cyclic oligoadenylates and subsequent allosteric activation of proteins that carry sensory domains is a distinctive feature of type III CRISPR-Cas systems. In this work, we characterize a set of associated genes of a type III-B system from Haliangium ochraceum that contains two caspase-like proteases, SAVED-CHAT and PCaspase (prokaryotic caspase), co-opted from a cyclic oligonucleotide-based antiphage signaling system (CBASS). Cyclic tri-adenosine monophosphate (AMP)-induced oligomerization of SAVED-CHAT activates proteolytic activity of the CHAT domains, which specifically cleave and activate PCaspase. Subsequently, activated PCaspase cleaves a multitude of proteins, which results in a strong interference phenotype in vivo in Escherichia coli. Taken together, our findings reveal how a CRISPR-Cas-based detection of a target RNA triggers a cascade of caspase-associated proteolytic activities.

Fig. 1.. Type III CRISPR-Cas operon associated SAVED-CHAT/PCaspase gene cluster setup and CBASS origin.

(A) Schematic representation of the H. ochraceum type III-B CRISPR-Cas operon containing the Cmr1–6 genes encoding the crRNA-guided type III protein complex and its associated genes: SAVED-CHAT, PC-σ, PCi, PCk and PCaspase (locus tags Hoch_1313–1323). (B) Domain architecture of SAVED-CHAT. (C) Domain architecture of PCaspase. (D) Genomic neighborhoods containing SAVED-CHAT and PCaspase. (E) SAVED domain phylogenetic midpoint-rooted tree. (F) CBASS type operons containing Haliangium SAVED clade domains with associated proteins containing cyclase, protein kinase, Sigma-70 and transposase domains.

Fig. 2.. cA₃-induced SAVED-CHAT and subsequent PCaspase activity.

(A) SDS-PAGE analysis of SAVED-CHAT cleavage, showing its dependency on cA₃ for cleaving PCaspase. (B) SDS-PAGE analysis of cleavage activity of activated SAVED-CHAT on PC-σ and PCi. (C) SDS-PAGE analysis of SAVED-CHAT cleavage on WT (wild-type) PCaspase and the R153A PCaspase mutant (D) SDS-PAGE analysis of cleavage activity of activated PCaspase on PC-σ and PCi. Activated PCaspase cleaves PC-σ and PCi individually, PC-σ is cleaved less when PCi is present. Stabilized PCaspase fragment is indicated with an asterix “*”.

Fig. 3.. PCaspase activity on non-associated substrates.

(A) SDS-PAGE analysis of the proteolytic activation of PCaspase by SAVED-CHAT, resulting in degradation of the casein substrate, in contrast to activated SAVED-CHAT by itself or the dPCaspase catalytic mutant (H79A, C146A). (B) Schematic overview of PCaspase activity assay using a fluorescent peptide reporter. (C) Increasing the molar ratio (PCi:PCaspase) of PCi reduces the cleavage activity of a FAM-peptide substrate by activated PCaspase. The faded colors represent the standard error of the mean (n=3). (D) Sensitivity for cA₃ of the FAM-peptide visualization method is 15.6 nM. The faded colors represent the standard error of the mean (technical replicates, n=3). (E) PCaspase cleaves reporter peptides containing arginines. Arginines are in red, fluorophores in green, and quencher in black. ν = norvaline, χ = cyclohexylalanine, δ = diaminobutyric acid, γ = GABA. The faded colors represent the standard error of the mean (n=3).

Fig 4.. PCaspase activation reduces the transformation efficiency of a target plasmid.

(A) Schematic overview of the experimental setup in E. coli BL21-AI. (B) Transformation efficiencies (relative to the non-target control) of target or non-target plasmid in E. coli co-expressing H. ochraceum type III CRISPR-Cas complex and different combinations of effector proteins. (C) Transformation efficiencies (relative to the non-target control) when SAVED-CHAT and PCaspase are co-expressed with PCi and PC-σ. Statistical significance was calculated using one-sided unpaired Welch’s t-test. * - p < 0.05, ** - p < 0.005, *** - p < 0.0005 (n=3).

Fig. 5.. Structural basis for SAVED-CHAT activation by cA₃.

(A) Representative 2D class averages of SAVED-CHAT bound to cA₃, showing side and top views. In the side view, the filament curvature is evident, with the CHAT domain at the periphery of the arch. In the top view, singlet filaments form a partial inter-filament doublet, with two singlets running in opposite polarities forming cross-fiber contacts spanning ~3 monomers. (B, C) 3.1 Å-resolution cryo-EM reconstruction and model of the cA₃-bound SAVED-CHAT filament. In some monomers, the CHAT domain is disordered and absent from the reconstruction (red and light green monomers). (D) Close-up view of the CHAT-CHAT singlet intra-filament interface, consisting of a four-helix bundle. (E) Close-up view of the CHAT-CHAT doublet inter-filament interface. (F) cA₃ binding site, at the interface between adjacent SAVED domains. Cryo-EM density for cA₃ is shown as translucent grey. (G) CHAT domain active site. The residues H375 and C422 that comprise catalytic dyad (magenta) are 3.8 Å apart and located within the substrate-binding channel beneath two unstructured gating loops (yellow). (H) Binding of cA3 to the SAVED domain of SAVED-CHAT induces oligomerization that activates its CHAT domain. Activated SAVED-CHAT cleaves PCaspase, which subsequently mediates further downstream events by cleaving PCi (inhibitor of PCaspase), PC-σ (transcriptional response?) and/or other essential host proteins. Hypothesized events are in grey.