Activation mechanism of a short argonaute-TIR prokaryotic immune system

Abstract

Short prokaryotic argonaute (pAgo) and toll/interleukin-1 receptor/resistance protein (TIR)–analog of PAZ (APAZ) form a heterodimeric SPARTA complex that provides immunity to its prokaryotic host through an abortive infection mechanism. Monomeric SPARTA senses foreign RNA/DNA duplexes to assemble an active tetramer resulting in cell death by nicotinamide adenine dinucleotide (oxidized form) (NAD) depletion via an unknown mechanism. We report nine structures of SPARTA in different functional states at a resolution range of 4.2 to 2.9 angstroms, revealing its activation mechanism. Inactive SPARTA monomers bind to RNA/DNA duplexes to form symmetric dimers mediated by the association of Ago subunits. The initiation of tetramer assembly induces flexibility of the TIR domains enabling a symmetry-breaking rotational movement of a TIR domain in the dimer units which facilitates the TIR oligomerization, resulting in the formation of the substrate binding pocket and the activation of the SPARTA complex’s NADase activity. Our findings provide detailed structural and mechanistic insights into activating a short argonaute defense system.

Target DNA-dependent activation mechanism of the prokaryotic immune system SPARTA

“…Beyond insights derived in our study, these studies reveal structures of SPARTA bound to the guide RNA-target DNA duplex prior to oligomerization ( 66 ) and in the dimeric form prior to tetramerization ( 60 , 64 , 67 ). Furthermore, mutational analyses in these studies extend insights into the structural determinants of guide RNA and target DNA binding ( 67 ), TIR-TIR interactions ( 60 , 61 , 66 , 67 ), and interactions of the TIR domains with the NAD substrate ( 61 , 62 ). Together with our structural analysis, these studies provide a comprehensive understanding of SPARTA mechanisms.…”

The missing part: the Archaeoglobus fulgidus Argonaute forms a functional heterodimer with an N-L1-L2 domain protein

“…Our structural and functional characterization of the restored full AfAgo complex coupled with computational analysis of its homologs indicates that long-B Agos can be divided into two major groups: typical single-chain pAgos exemplified by RsAgo, and split pAgos, exemplified by full AfAgo. Full AfAgo and other split long-B Agos are similar to short pAgos, which also assemble into functional heterodimeric complexes composed of an APAZ-containing protein (a structural equivalent of the N-L1-L2 domains ( 9 , 10 , 11 , 12 )) and a MID-PIWI protein. The major difference between the short and fAfAgo-like split pAgo systems is that the N1-L1-L2 subunit in short pAgo systems is often fused to an effector domain.…”

“…The majority (∼60%) of pAgos belong to the group of highly divergent short pAgos, which contain only the MID and inactive PIWI domains. In their operons or polypeptides, short pAgos are typically associated with APAZ (Analog of PAZ) domain-containing effector proteins that according to structure modelling and experimental structures correspond to the N, L1 and L2 domains of long pAgos ( 8 , 9 , 10 , 11 , 12 ). APAZ-containing proteins are often fused to effector Sir2 (Silent informator regulator 2), Mrr nucleases or TIR (Toll-Interleukin-1 Receptor) domains.…”

Structural basis of antiphage immunity generated by a prokaryotic Argonaute-associated SPARSA system

“…The mechanism of SPARTA NADase activation has been reported recently and depends on the tetramerization of the TIR domain mediated by the MID:MID interaction of pAgo 47 – 50 . Tetramerization of SPARTA, which is induced by the RNA/DNA duplex, promotes the formation of the substrate binding pocket mediated by the BB loop and EE surface, thereby triggering the SPARTA complex’s NADase activity 43 , 47 , 49 , 51 , 52 .…”