CRISPR antiphage defence mediated by the cyclic nucleotide-binding membrane protein Csx23

Abstract CRISPR-Cas provides adaptive immunity in prokaryotes. Type III CRISPR systems detect invading RNA and activate the catalytic Cas10 subunit, which generates a range of nucleotide second messengers to signal infection. These molecules bind and activate a diverse range of effector proteins that provide immunity by degrading viral components and/or by disturbing key aspects of cellular metabolism to slow down viral replication. Here, we focus on the uncharacterised effector Csx23, which is widespread in Vibrio cholerae. Csx23 provides immunity against plasmids and phage when expressed in Escherichia coli along with its cognate type III CRISPR system. The Csx23 protein localises in the membrane using an N-terminal transmembrane α-helical domain and has a cytoplasmic C-terminal domain that binds cyclic tetra-adenylate (cA4), activating its defence function. Structural studies reveal a tetrameric structure with a novel fold that binds cA4 specifically. Using pulse EPR, we demonstrate that cA4 binding to the cytoplasmic domain of Csx23 results in a major perturbation of the transmembrane domain, consistent with the opening of a pore and/or disruption of membrane integrity. This work reveals a new class of cyclic nucleotide binding protein and provides key mechanistic detail on a membrane-associated CRISPR effector.

A: Plasmid challenge assay, demonstrating that all variants retained biological function.

Figure S1 .
Figure S1.Distance tree of PSI-BLAST results using Csx23 (WP_001091334.1, highlighted) as query (NCBI website).The tree is based on pairwise alignments with fast minimum evolution (Grishin distance model, 0.85 maximum sequence difference) as method for tree construction.

P 2 Figure S2 .BFigure S3 .
Figure S2.In silico analyses of Csx23.A: Predicted Aligned Error (PAE) plot output from AF2 for FL Csx23.The colour at (x, y) indicates the expected position error at residue x (x axis) if the predicted and true structures were aligned on residue y (y axis).Coloured from blue (low error) to red (high error) as indicated on side bar in Å.This plot shows the residue positional predictions within each domain have low error, but predictions of the residue positions between the two domains is higher.B: Prediction of transmembrane domains using DeepTMHMM indicating very strongly a membrane-spanning N-terminal domain and a soluble, cytosolic C-terminal domain.

Figure S6 .Figure S7 .
Figure S6.Comparison of the structures of Csx23 CTD and the PB1 domain from protein kinase C zeta type.A: Cartoon representation of a single subunit of Csx23 CTD coloured from N-(blue) to C-terminus (red).B: Cartoon representation of the PB1 domain from protein kinase C zeta type from rat (PDB: 4MJS) coloured from N-(blue) to C-terminus (red).C: Superimposition of Csx23 CTD (green cartoon) and the PB1 domain from protein kinase C zeta type (light blue cartoon).D: Superimposition of Csx23 CTD (green cartoon) and the PB1 domain from protein kinase C zeta type (light blue cartoon), highlighting a conserved lysine residue at the end of the first beta-strand in each protein, and the additional region containing acidic residues present only in the PB1 domain.

Figure S8 .
Figure S8.Characterisation of Csx23 variants used for spin-labeling and EPR experiments.

FigureFigure S9 .
Figure representative of two technical replicates.B: SDS-PAGE of purified variants of Csx23.

Figure S10 .
Figure S10.PELDOR data for the Csx23 AALA V52R1 mutant in absence (left) and presence (right) of cyclic nucleotide (cA4), comparing micellar protein (detergent) to protein reconstituted in nanodiscs (ND).The predicted distribution based on the AF2 tetramer predicted structure of Csx23 is shown in blue.

Figure S11 .
Figure S11.PELDOR data for the Csx23 AALA N59R1 mutant reconstituted in nanodiscs (ND) in presence (red) and absence (grey) of cyclic nucleotide (cA4).Raw PELDOR data (top left) and background-corrected traces with fits (top right); overlay of corresponding distance distributions shown as 95% confidence bands with predicted distributions from MMM and mtsslWizard based on AF2 predicted structure (bottom left), colour bars indicate reliability ranges (green: shape reliable; yellow: mean and width reliable; orange: mean reliable; red: no quantification possible); cartoon representation of AF2 predicted tetrameric structure of the spin-labelled Csx23 AALA N59R1 tetramer (bottom right).

Figure S12 .
Figure S12.PELDOR data for the Csx23 AALA N62R1 mutant reconstituted in nanodiscs (ND) in presence (red) and absence (grey) of cyclic nucleotide (cA4).Raw PELDOR data (top left) and background-corrected traces with fits (top right); overlay of corresponding distance distributions shown as 95% confidence bands with predicted distributions from MMM and mtsslWizard based on AF2 structure (bottom left), colour bars indicate reliability ranges (green: shape reliable; yellow: mean and width reliable; orange: mean reliable; red: no quantification possible); cartoon representation of AF2 predicted tetrameric structure of the spin-labelled Csx23 AALA N62R1 tetramer (bottom right).

Figure S13 .
Figure S13.Phage P1 immunity assay using the VmeCmr / effector system.The growth of cells harbouring either Csx23 or NucC in the absence of targeting crRNA (dashed, salmon line) is the same as that of cells not carrying an effector (solid, grey line).The combination of targeting crRNA and effector results (solid, salmon line) in significantly faster recovery of the culture at high MOIs (MOI 15).

Table S1 .
VmeCRISPR repeat and spacer sequences used in this study.

Table S3 .
Data collection and refinement statistics for the structure of Csx23 CTD in complex with cA4.
* Values in parentheses correspond to data in the high resolution shell ** RMSD, root mean square deviation