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bioRxiv · 10.1101/2023.09.17.558117

Nucleic Acid Mediated Activation of a Short Prokaryotic Argonaute Immune System

Aggarwal, A. K.·Kottur, J.·Malik, R.
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Abstract

The continual pressure of invading DNA has led bacteria to develop numerous immune systems, including a short prokaryotic Argonaute (pAgo) TIR-APAZ system (SPARTA) that is activated by invading DNA to unleash its TIR domain for NAD(P)+ hydrolysis. To gain a molecular understanding of this activation process, we resolved a crystal structure of SPARTA heterodimer in the absence of guide RNA/target ssDNA at 2.66[A] resolution and a cryo-EM structure of the SPARTA oligomer (tetramer of heterodimers) bound to guide RNA/target ssDNA at nominal 3.15-3.35[A] resolution. The crystal structure provides a high-resolution view of the TIR-APAZ protein and the MID-PIWI domains of short pAgo - wherein, the APAZ domain emerges as equivalent to the N, L1 and L2 regions of long pAgos and the MID domain has a unique insertion (insert57). A comparison to cryo-EM structure reveals regions of the PIWI (loop10-9) and APAZ (helix N) domains that reconfigure to relieve auto-inhibition to permit nucleic acid binding and transition to an active oligomer. Oligomerization is accompanied by the nucleation of the TIR domains in a parallel-strands arrangement for catalysis. Together, the structures provide a visualization of SPARTA before and after RNA/ssDNA binding and reveal the basis of SPARTAs active assembly leading to NAD(P)+ degradation and abortive infection.

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Aggarwal, A. K., Kottur, J., Malik, R.. 2023-09-17. Nucleic Acid Mediated Activation of a Short Prokaryotic Argonaute Immune System. https://doi.org/10.1101/2023.09.17.558117

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