Oligomerization-mediated activation of a short prokaryotic Argonaute

Although eukaryotic and long prokaryotic Argonaute proteins (pAgos) cleave nucleic acids, some short pAgos lack nuclease activity and hydrolyse NAD(P)+ to induce bacterial cell death1. Here we present a hierarchical activation pathway for SPARTA, a short pAgo consisting of an Argonaute (Ago) protein and TIR–APAZ, an associated protein2. SPARTA progresses through distinct oligomeric forms, including a monomeric apo state, a monomeric RNA–DNA-bound state, two dimeric RNA–DNA-bound states and a tetrameric RNA–DNA-bound active state. These snapshots together identify oligomerization as a mechanistic principle of SPARTA activation. The RNA–DNA-binding channel of apo inactive SPARTA is occupied by an auto-inhibitory motif in TIR–APAZ. After the binding of RNA–DNA, SPARTA transitions from a monomer to a symmetric dimer and then an asymmetric dimer, in which two TIR domains interact through charge and shape complementarity. Next, two dimers assemble into a tetramer with a central TIR cluster responsible for hydrolysing NAD(P)+. In addition, we observe unique features of interactions between SPARTA and RNA–DNA, including competition between the DNA 3′ end and the auto-inhibitory motif, interactions between the RNA G2 nucleotide and Ago, and splaying of the RNA–DNA duplex by two loops exclusive to short pAgos. Together, our findings provide a mechanistic basis for the activation of short pAgos, a large section of the Ago superfamily.