6ra4
From Proteopedia
Human ARGONAUTE-2 PAZ DOMAIN (214-347) IN COMPLEX WITH CGUGACUCU
Structural highlights
Function[AGO2_HUMAN] Required for RNA-mediated gene silencing (RNAi) by the RNA-induced silencing complex (RISC). The 'minimal RISC' appears to include EIF2C2/AGO2 bound to a short guide RNA such as a microRNA (miRNA) or short interfering RNA (siRNA). These guide RNAs direct RISC to complementary mRNAs that are targets for RISC-mediated gene silencing. The precise mechanism of gene silencing depends on the degree of complementarity between the miRNA or siRNA and its target. Binding of RISC to a perfectly complementary mRNA generally results in silencing due to endonucleolytic cleavage of the mRNA specifically by EIF2C2/AGO2. Binding of RISC to a partially complementary mRNA results in silencing through inhibition of translation, and this is independent of endonuclease activity. May inhibit translation initiation by binding to the 7-methylguanosine cap, thereby preventing the recruitment of the translation initiation factor eIF4-E. May also inhibit translation initiation via interaction with EIF6, which itself binds to the 60S ribosomal subunit and prevents its association with the 40S ribosomal subunit. The inhibition of translational initiation leads to the accumulation of the affected mRNA in cytoplasmic processing bodies (P-bodies), where mRNA degradation may subsequently occur. In some cases RISC-mediated translational repression is also observed for miRNAs that perfectly match the 3' untranslated region (3'-UTR). Can also up-regulate the translation of specific mRNAs under certain growth conditions. Binds to the AU element of the 3'-UTR of the TNF (TNF-alpha) mRNA and up-regulates translation under conditions of serum starvation. Also required for transcriptional gene silencing (TGS), in which short RNAs known as antigene RNAs or agRNAs direct the transcriptional repression of complementary promoter regions.[1] [2] [3] [4] [5] [6] [7] [8] [9] [10] [11] [12] [13] [14] [15] [16] [17] [18] [19] [20] [21] [22] Publication Abstract from PubMedThe Argonaute-2 protein is part of the RNA-induced silencing complex (RISC) and anchors the guide strand of the small interfering RNA (siRNA). The 3' end of the RNA contains two unpaired nucleotides (3'-overhang) that interact with the PAZ (PIWI-Argonaute-Zwille) domain of the protein. Theoretical and experimental evidence points towards a direct connection between the PAZ/3'-overhang binding affinity and siRNA's potency and specificity. Among the challenges to overcome when deploying siRNA molecules as therapeutics are their ready degradation under physiological conditions, and off-target effects. It has been demonstrated that nuclease resistance can be improved via replacement of the dinucleotide overhang by small molecules which retain the interactions of the RNA guide strand with the PAZ domain. Most commonly, nucleotide analogues are used to substitute the siRNA overhang. However, in this study we adopt a de novo approach to its modification. The X-ray structure of human Argonaute-2 PAZ domain served to perform virtual screening and molecular interaction energy profiling (i.e., decomposition of the force field calculated protein-ligand interaction energies) of tailored-to-purpose fragment libraries. The binding of fragments to the PAZ domain was validated experimentally by NMR spectroscopy. The in silico guided protocol led to the efficient discovery of a number of PAZ domain ligands with affinities comparable to that of a reference dinucleotide (UpU, Kd = 33 microM). Originally starting from a generic fragment library, hits progress from 930 microM down to 14 microM within 3 iterations for the fragments selected via in silico molecular interaction energy profiling from a bespoke library. These dinucleotide siRNA guide strand surrogates represent potential new siRNA-based therapeutics (when attached to siRNA to form bioconjugates) featuring improved efficacy, specificity, stability and cellular uptake. This project yielded a portfolio of 7 patent applications, two of which have been granted to date. How to Computationally Stack the Deck for Hit-to-Lead Generation: In Silico Molecular Interaction Energy Profiling for De Novo siRNA Guide Strand Surrogate Selection.,Greenidge P, Blommers MJJ, Priestle JP, Hunziker J J Chem Inf Model. 2019 Apr 25. doi: 10.1021/acs.jcim.8b00892. PMID:31021613[23] From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine. Loading citation details.. Citations 1 reviews cite this structure No citations found References
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