Structural Biology Program, Memorial Sloan-Kettering Cancer Center, New York, NY 10065, USA.
Cell Rep. 2013 Jun 27;3(6):1893-900. doi: 10.1016/j.celrep.2013.06.010.
We have solved the crystal structure of human ARGONAUTE1 (hAGO1) bound to endogenous 5'-phosphorylated guide RNAs. To identify changes that evolutionarily rendered hAGO1 inactive, we compared our structure with guide-RNA-containing and cleavage-active hAGO2. Aside from mutation of a catalytic tetrad residue, proline residues at positions 670 and 675 in hAGO1 introduce a kink in the cS7 loop, forming a convex surface within the hAGO1 nucleic-acid-binding channel near the inactive catalytic site. We predicted that even upon restoration of the catalytic tetrad, hAGO1-cS7 sterically hinders the placement of a fully paired guide-target RNA duplex into the endonuclease active site. Consistent with this hypothesis, reconstitution of the catalytic tetrad with R805H led to low-level hAGO1 cleavage activity, whereas combining R805H with cS7 substitutions P670S and P675Q substantially augmented hAGO1 activity. Evolutionary amino acid changes to hAGO1 were readily reversible, suggesting that loading of guide RNA and pairing of seed-based miRNA and target RNA constrain its sequence drift.
我们已经解析了与人源 ARGONAUTE1（hAGO1）结合的内源性 5'-磷酸化指导 RNA 的晶体结构。为了鉴定使 hAGO1 失活的进化过程中的变化，我们将我们的结构与含有指导 RNA 且具有切割活性的 hAGO2 进行了比较。除了催化四联体残基的突变外，hAGO1 中的脯氨酸残基 670 和 675 会使 cS7 环发生扭曲，在靠近无活性催化位点的 hAGO1 核酸结合通道内形成凸面。我们预测，即使恢复了催化四联体，hAGO1-cS7 也会阻碍完全配对的指导靶 RNA 双链进入内切酶活性位点。与该假设一致，用 R805H 重建催化四联体导致 hAGO1 的低水平切割活性，而将 R805H 与 cS7 取代 P670S 和 P675Q 相结合则大大提高了 hAGO1 的活性。hAGO1 的进化氨基酸变化很容易是可逆的，这表明指导 RNA 的加载和基于种子的 miRNA 和靶 RNA 的配对限制了其序列漂移。
