Abstract：
The middle (MID) domain of eukaryotic Argonaute (Ago) proteins and archaeal and bacterial homologues mediates the interaction with the 5\'-terminal nucleotide of miRNA and siRNA guide strands. The MID domain of human Ago2 (hAgo2) is comprised of 139 amino acids with a molecular weight of 15.56 kDa. MID adopts a Rossman-like beta1-alpha1-beta2-alpha2-beta3-alpha3-beta4-alpha4 fold with a nucleotide specificity loop between beta3 and alpha3. Multiple crystal structures of nucleotides bound to hAgo2 MID have been reported, whereby complexes were obtained by soaking ligands into crystals of MID domain alone. This protocol describes a simplified one-step approach to grow well-diffracting crystals of hAgo2 MID-nucleotide complexes by mixing purified His6-SUMO-MID fusion protein, Ulp1 protease, and excess nucleotide in the presence of buffer and precipitant. The crystal structures of MID complexes with UMP, UTP and 2\'-3\' linked α-L-threofuranosyl thymidine-3\'-triphosphate (tTTP) are presented. This article also describes fluorescence-based assays to measure dissociation constants (Kd) of MID-nucleotide interactions for nucleoside 5\'-monophosphates and nucleoside 3\',5\'-bisphosphates. © 2024 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Crystallization of Ago2 MID-nucleotide complexes Basic Protocol 2: Measurement of dissociation constant Kd between Ago2 MID and nucleotides.
摘要：
真核Argonaute（Ago）蛋白以及古细菌和细菌同源物的中间（MID）结构域介导与miRNA和siRNA引导链的5'末端核苷酸的相互作用。人Ago2(hAgo2)的MID结构域由分子量为15.56kDa的139个氨基酸组成。MID采用Rossman样的β1-α1-β2-α2-β3-α3-β4-α4折叠，在β3和α3之间具有核苷酸特异性环。已经报道了与hAgo2MID结合的核苷酸的多个晶体结构，通过将配体单独浸入MID结构域的晶体中获得复合物。该协议描述了一种简化的一步方法，通过混合纯化的His6-SUMO-MID融合蛋白来生长hAgo2MID-核苷酸复合物的衍射良好的晶体，Ulp1蛋白酶，以及在缓冲液和沉淀剂存在下的过量核苷酸。MID与UMP配合物的晶体结构，介绍了UTP和2'-3'连接的α-L-苏呋喃基胸苷-3'-三磷酸(tTTP)。本文还介绍了基于荧光的测定法，以测量核苷5'-单磷酸和核苷3'的MID-核苷酸相互作用的解离常数（Kd），5\'-双磷酸盐。©2024作者WileyPeriodicalsLLC出版的当前协议。基本方案1:Ago2MID-核苷酸复合物的结晶基本方案2:Ago2MID和核苷酸之间的解离常数Kd的测量。
