Functionally comparable but evolutionarily distinct nucleotide-targeting effectors help identify conserved paradigms across diverse immune systems

Abstract

While nucleic acid-targeting effectors are known to be central to biological conflicts and anti-selfish element immunity, recent findings have revealed immune effectors that target their building blocks and the cellular energy currency-free nucleotides. Through comparative genomics and sequence-structure analysis, we identified several distinct effector domains, which we named Calcineurin-CE, HD-CE, and PRTase-CE. These domains, along with specific versions of the ParB and MazG domains, are widely present in diverse prokaryotic immune systems and are predicted to degrade nucleotides by targeting phosphate or glycosidic linkages. Our findings unveil multiple potential immune systems associated with at least 17 different functional themes featuring these effectors. Some of these systems sense modified DNA/nucleotides from phages or operate downstream of novel enzymes generating signaling nucleotides. We also uncovered a class of systems utilizing HSP90- and HSP70-related modules as analogs of STAND and GTPase domains that are coupled to these nucleotide-targeting- or proteolysis-induced complex-forming effectors. While widespread in bacteria, only a limited subset of nucleotide-targeting effectors was integrated into eukaryotic immune systems, suggesting barriers to interoperability across subcellular contexts. This work establishes nucleotide-degrading effectors as an emerging immune paradigm and traces their origins back to homologous domains in housekeeping systems.

Graphical Abstract

Figure 1.

Nucleotide-targeting effector reaction diversity. Schematic representations of reactions catalyzed by known and newly predicted effectors. Reactions proposed for the first time in this research are denoted by a blue circle next to the predicted enzyme. Reactions are thematically grouped into (A) NADase, (B) diverse nucleotide hydrolase, (C) base removal, (D) base modification and (E) nucleotide phosphorolysis reactions.

Figure 2.

(A) Previous characterized conflict systems containing Calcineurin-CE as an effector. Architectures and gene-neighborhoods of previously described conflict systems containing the calcineurin-CE domain as an effector. (B) Contextual network of different conflict systems containing the effectors characterized in the present work. Nodes with black outlines represent the three nucleotide-targeting domains characterized in the present work. Other nodes represent different immune systems named by the main domain of the given system. The coloring of these nodes corresponds to their presence in representative architectures/operons shown in panel A. Edges represent the presence of one of the nucleotide-targeting effectors in the connected system. Hexagonal nodes represent ATPases domains and rounded hexagonal nodes represent P-loop NTPases.

Figure 3.

Evolutionary analysis and structural features of the nucleotide-targeting conflict effectors. (A–C) (top) Phylogenetic trees of the three nucleotide-targeting effectors. The trees were constructed with IQtree2 using ultrafast bootstrap and SH-aLRT test as support values. The red circles in the branches indicate SH-aLRT ≥80% and UFboot ≥95% of support values. The clades of each effector are labeled according to the associated domains of the systems to which they belong. (A–C) (bottom) Structural models for representatives of each of the three domains were generated using Alphafold2. Residues synapomorphic to the conflict versions are shown in red, while those generally conserved in the superfamily are colored black.

Figure 4.

Domain architectures, gene-neighborhoods and structural models of systems with potential determinants for localization to specific macromolecular complexes. All structural models were generated using Alphafold2. (A) Systems with ORC/Cdc6 ATPase and double CARF proteins. Nus.Kinase: Nucleoside kinase. (B) Model of the hetero-multimer formed by the ORC/Cdc6, double CARF and Calcineurin-CE proteins: the molecular surface representation is shown above and the cartoon representation below. (C) Systems containing the PUA-like domain and predicted to sense modified viral DNA. (D) Model of PUA-like domain with inferred topology on the left and cartoon representation on the right. The arrow indicates the predicted modified nucleic-acid-binding cleft. (E) Alternative GreA/B-C systems predicted to localize to transcriptional complexes. (F) NAD⁺ derivative sensing systems. (G) Model of inactive Sirtuin domain showing retention of core structure despite loss of the catalytic residues. (H) Retron systems.

Figure 5.

Domain architectures, gene-neighborhoods and structural models of systems with simple themes and those with diverse sensory and signaling modules. All structural models were generated using Alphafold2. (A) Systems containing the ISOCOT domain and predicted to sense AdoMet derivatives. (B) Simple systems coupling two nucleotide-targeting effectors. (C) Systems coupling nucleotide-degrading effectors with inactive and/or active thymidylate synthase domains. (D) Systems with the ParB-CE and thymidylate synthase/kinase domains. (E) ParB-CE with the AEPrimase-1D and CCA-adding enzyme (CCAA)-like Pol-β-based systems. (F) Histidine kinase signaling combined with second messenger systems. (G) Model of PRTase domains from the above systems showing their peculiar predicted Zn-binding hood region. (H) Novel STAND-based systems. (I) AP-GTPase systems. (J) MNS-type STAND systems. (K) AP-ATPase systems. (L) Systems containing dynamin-like domains. (M, N) NACHT systems. (O) NACHT systems with inactivated Calcineurin. β-lect: β-sandwich lectin.

Figure 6.

Domain architectures, gene-neighborhoods and structural models of systems with HSP90, HSP70 and Pepco domains (A) HSP90-based systems. F.E.: Fast-evolving domain. (B) Alphafold2 model of an exemplar of the HSP90 system with C-terminal wHTH domains forming a toroidal structure; the molecular surface representation is shown above and the cartoon representation below. (C) HSP70-based systems. (D) Pepco-peptidase-containing systems. (E) Multimer model of the Pepco (top) and a caspase domain (bottom). The catalytic histidine and cysteine of caspase are shown along with the conserved aspartate in the unstructured region of the Pepco domain. (F) Multimer model of the Pepco octamer forming a toroid.