Crystal structure and ligand binding of the MID domain of a eukaryotic Argonaute protein

Department of Biochemistry, Max Planck Institute for Developmental Biology, Spemannstrasse 35, Tübingen D-72076, Germany.
EMBO Reports (Impact Factor: 9.06). 07/2010; 11(7):522-7. DOI: 10.1038/embor.2010.81
Source: PubMed


Argonaute (AGO) proteins are core components of RNA-induced silencing complexes and have essential roles in RNA-mediated gene silencing. They are characterized by a bilobal architecture, consisting of one lobe containing the amino-terminal and PAZ domains and another containing the MID and PIWI domains. Except for the PAZ domain, structural information on eukaryotic AGO domains is not yet available. In this study, we report the crystal structure of the MID domain of the eukaryotic AGO protein QDE-2 from Neurospora crassa. This domain adopts a Rossmann-like fold and recognizes the 5'-terminal nucleotide of a guide RNA in a manner similar to its prokaryotic counterparts. The 5'-nucleotide-binding site shares common residues with a second, adjacent ligand-binding site, suggesting a mechanism for the cooperative binding of ligands to the MID domain of eukaryotic AGOs.

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Available from: Andreas Boland, Aug 21, 2014
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    • "Guided by miRNAs, AGO proteins recognize target genes at complementary sites to repress gene translation by cleaving target mRNAs meanwhile binding to cap structure of mRNAs, and in some cases to repress gene transcription by RNA directed DNA methylation [10-14]. Typical AGO protein contains a variable N-terminal domain, a conserved C-terminal PAZ domain that recognizes the 3′ end of small RNAs, a MID (middle) that binds to the 5′ phosphate of small RNAs and a PIWI domain carrying an Asp-Asp-His (DDH) motif as an active site which exhibits endonuclease activity similar to that of RNaseH [6,7,15-17]. "
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    ABSTRACT: AGO (Argonaute) protein participates in plant developmental processes and virus defense as a core element of transcriptional regulator or/and post-transcriptional regulator in RNA induced silencing complex (RISC), which is guided by small RNAs to repress target genes expression. Previously, it was revealed that 15 putative AGO genes in tomato genome. In present study, out of 15 detected SlAGO genes, only SlAGO4C and SlAGO15 couldn't be detected in roots, stems, leaves, buds, flowers and fruit of tomato by 30 cycles of PCR. SlAGO7 could be detected in early stage of fruit (-2 dpa, 0 dpa and 4 dpa), but it was significantly down-regulated in fruit collected on the 6 days post anthesis. Moreover, SlAGO5 could only be detected in reproductive tissues and SlAGO4D was specifically detected in fruit. According to blast result with miRNA database, three SlAGO genes harbored complementary sequences to miR168 (SlAGO1A and SlAGO1B) or miR403 (SlAGO2A). 5[prime] RACE (Rapid amplification of cDNA ends) mapping was used to detect the 3[prime] cleavage products of SlAGO mRNAs. In addition, subcellular localization of SlAGO proteins was detected. Our results showed that most SlAGO proteins localized to nucleus and cytoplasm. Importantly, nuclear membrane localization of AGO proteins was observed. Furthermore, mutated miR168 complementary site of SlAGO1A resulted in expanded localization of SlAGO1A, indicating that miR168 regulated localization of SlAGO1A. Our results contribute to demonstration of potential roles of these newly isolated AGO family in tomato developmental processes and proved the conserved relationships between AGO genes and miRNAs in tomato, which might play important roles in tomato development and virus defense.
    Full-text · Article · Sep 2013 · BMC Plant Biology
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    • "MID domain is involved in binding the 5' phosphate of siRNA [13], while PAZ domain is important for binding the 3' end of siRNA [14], [15]. The binding properties of MID domain are less dynamically variable than PAZ domain, thus underscoring its vulnerability for siRNA modifications [16], [17]. The events occurring at the binding of 3' nucleotide of siRNA with PAZ involve a series of interesting molecular dynamics during siRNA-Agos binding and cleavage mechanisms. "
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    ABSTRACT: RNA interference (RNAi) is a highly specialized process of protein-siRNA interaction that results in the regulation of gene expression and cleavage of target mRNA. The PAZ domain of the Argonaute proteins binds to the 3' end of siRNA, and during RNAi the attaching end of the siRNA switches between binding and release from its binding pocket. This biphasic interaction of the 3' end of siRNA with the PAZ domain is essential for RNAi activity; however, it remains unclear whether stronger or weaker binding with PAZ domain will facilitate or hinder the overall RNAi process. Here we report the correlation between the binding of modified siRNA 3' overhang analogues and their in vivo RNAi efficacy. We found that higher RNAi efficacy was associated with the parameters of lower Ki value, lower total intermolecular energy, lower free energy, higher hydrogen bonding, smaller total surface of interaction and fewer van der Waals interactions. Electrostatic interaction was a minor contributor to compounds recognition, underscoring the presence of phosphate groups in the modified analogues. Thus, compounds with lower binding affinity are associated with better gene silencing. Lower binding strength along with the smaller interaction surface, higher hydrogen bonding and fewer van der Waals interactions were among the markers for favorable RNAi activity. Within the measured parameters, the interaction surface, van der Waals interactions and inhibition constant showed a statistically significant correlation with measured RNAi efficacy. The considerations provided in this report will be helpful in the design of new compounds with better gene silencing ability.
    Full-text · Article · Feb 2013 · PLoS ONE
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    • "Except for the 5 0 -phosphate binding pocket for siRNA or miRNA, Djuranovic et al. [2010] proposed that Dm_AGO1 has a second miRNA-dependent site that can bind nucleotides such as the 5 0 -cap. The second ligand-binding site of Dm_AGO1 might be under allosteric control, whereas the other AGO proteins might be regulated by distinct ligand or might no longer be regulated by a second ligand [Boland et al., 2010]. According to this, it induces us to speculate that the second sulfate ion in Nc_QDE2 MID domain may occupy the second ligand binding site. "
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    ABSTRACT: Argonaute (AGO) proteins are highly specialized small-RNA-binding modules and small RNAs are anchored to their specific binding pockets guiding AGO proteins to target mRNA molecules for silencing or destruction. The 135 full-length AGO protein sequences derived from 36 species covering prokaryote, archaea, and eukaryote are chosen for structural and functional analyses. The results show that bacteria and archaeal AGO proteins are clustered in the same clade and there exist multiple AGO proteins in most eukaryotic species, demonstrating that the increase of AGO gene copy number and horizontal gene transfer (HGT) have been the main evolutionary driving forces for adaptability and biodiversity. And the emergence of PAZ domain in AGO proteins is the unique evolutionary event. The analysis of middle domain (MID)-nucleotide contaction shows that either the position of sulfate I bond in Nc_QDE2 or the site of phosphate I bond in Hs_AGO2 represents the 5'-nucleotide binding site of miRNA. Also, H334, T335, and Y336 of Hs_AGO1 can form hydrogen bonds with 3'-overhanging ends of miRNAs and the same situation exists in Hs_AGO2, Hs_AGO3, Hs_AGO4, Dm_AGO1, and Ce_Alg1. Some PIWI domains containing conserved DDH motif have no slicer activity, and post-translational modifications may be associated with the endonucleolytic activities of AGOs. With the numbers of AGO genes increasing and fewer crystal structures available, the evolutionary and functional analyses of AGO proteins can help clarify the molecular mechanism of function diversification in response to environmental changes, and solve major issues including host defense mechanism against virus infection and molecular basis of disease.
    Full-text · Article · Aug 2012 · Journal of Cellular Biochemistry
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