Prokaryotic Argonaute fromArchaeoglobus fulgidusinteracts with DNA as a homodimer

Abstract

14Background: Argonaute (Ago) proteins are found in all three domains of life. The best characterized 15 group is eukaryotic Argonautes (eAgos), which are the core of RNA interference. The best understood 16 prokaryotic Ago (pAgo) proteins are full-length pAgos. They are monomeric proteins, all composed of 17 four major structural/functional domains (N, PAZ, MID and PIWI) and thereby closely resemble eAgos. 18It is believed that full-length pAgos function as prokaryotic antiviral systems, with the PIWI domain 19 performing cleavage of invading nucleic acids. However, the majority of identified pAgos are shorter 20 and catalytically inactive (encode just MID and inactive PIWI domains), thus their action mechanism 21 and function remain unknown. 22Results: In this work we focus on AfAgo, a short pAgo protein encoded by an archaeon 23 Archaeoglobus fulgidus. We find that in all previously solved AfAgo structures, its two monomers form 24 substantial dimerization interfaces involving the C-terminal β-sheets. Led by this finding, we have 25 employed various biochemical and biophysical assays, including single-molecule FRET, SAXS and 26 AFM, to test the possible dimerization of AfAgo. SAXS results confirm that WT AfAgo, but not the 27 dimerization surface mutant AfAgoΔ, forms a homodimer both in the apo-form and when bound to a 28 nucleic acid. Single molecule FRET and AFM studies demonstrate that the dimeric WT AfAgo binds 29 two ends of a linear DNA fragment, forming a relatively stable DNA loop. 30Conclusion: Our results show that contrary to other characterized Ago proteins, AfAgo is a stable 31 homodimer in solution, which is capable of simultaneous interaction with two DNA molecules. This 32 finding broadens the range of currently known Argonaute-nucleic acid interaction mechanisms. Argonaute (Ago) proteins are found in all three domains of life (bacteria, archaea, and eukaryotes). 37The best characterized group is eukaryotic Ago (eAgo) proteins. Being the functional core of RNA 38 interference machinery, eAgos are involved in regulation of gene expression, silencing of mobile 39 genome elements, and defense against viruses. From the structural and mechanistic point of view, all 40 eAgos are very similar, as they all use small RNA molecules as guides for sequence-specific 41 recognition of RNA targets, and are monomeric proteins sharing four conserved functional domains, 42 which are organized in a bilobed structure [1]. The N-terminal lobe consists of the N-domain that 43 separates guide and target strands [2], and the PAZ domain responsible for binding the 3′-terminus of 44 the guide RNA; the C-terminal lobe consists of the MID domain, which binds the 5′-terminus of the 45 guide RNA, and the PIWI domain, an RNase. Upon recognition of the RNA target, eAgos may either 46 cleave it employing the catalytic activity of the PIWI domain, or, especially eAgo proteins that encode 47 catalytically inactive PIWI domains, recruit partner proteins leading to degradation of the target RNA 48 or repression of its translation ...