Poster abstracts
Poster number 152 submitted by Xiaoyuan Yang
Oligomerization-mediated activation of a short prokaryotic Argonaute
Xiaoyuan Yang (Department of Biological Chemistry and Pharmacology, The Ohio State University ), Zhangfei Shen (Department of Biological Chemistry and Pharmacology, The Ohio State University), Shiyu Xia (Divison of Biology and Biological Engineering, California Institute of Technology), Wei Huang, Derek J. Taylor (Department of Biochemistry, Case Western Reserve University School of Medicine), Kotaro Nakanishi (Department of Chemistry and Biochemistry, The Ohio State University), Tianmin Fu (Department of Biological Chemistry and Pharmacology, The Ohio State University)
Abstract:
Argonautes (Agos) are highly conserved proteins that widely exist in all domains of life, and they play a crucial role in post-transcriptional gene regulation and immune surveillance against foreign invaders. While eukaryotic Agos (eAgos) and long prokaryotic Agos (pAgos) employ short nucleotide guides to cleave complementary target nucleic acids, short pAgos lack such cleavage activity and are usually associated with distinct accessory proteins for function. However, compared to the well-studied eAgos and long pAgos, the short pAgos, which constitute the majority of pAgos, remain underexplored. Here, we present a hierarchical activation pathway of a short pAgo system named SPARTA (Short Prokaryotic Argonautes and associated TIR-APAZ proteins). 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. Apo SPARTA is inactive, its RNA-DNA-binding channel occupied an auto-inhibitory motif in TIR-APAZ. Upon RNA-DNA binding, SPARTA transitions from a monomer to a symmetric and then an asymmetric dimer, in which two TIR domains interact via charge and shape complementarity. Next, two dimers assemble into a tetramer with a central TIR cluster responsible for hydrolyzing NAD(P)+. Additionally, we observed unique features of SPARTA-RNA-DNA interactions, 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 pAgo systems and offer insights into repurposing SPARTA as valuable tools for cancer diagnostics and therapy.
References:
1. Koopal, B., Potocnik, A., Mutte, S. K., Aparicio-Maldonado, C., Lindhoud, S., Vervoort, J. J. M., Brouns, S. J. J., & Swarts, D. C. (2022). Short prokaryotic Argonaute systems trigger cell death upon detection of invading DNA. Cell, 185(9), 1471–1486.e19. https://doi.org/10.1016/j.cell.2022.03.012
2. Shen, Z., Yang, XY., Xia, S. et al. Oligomerization-mediated activation of a short prokaryotic Argonaute. Nature 621, 154–161 (2023). https://doi.org/10.1038/s41586-023-06456-z
Keywords: Short Prokaryotic Argonautes, Oligomerization-mediated activation