Markov State Models Reveal a Two-Step Mechanism of miRNA Loading into the Human Argonaute Protein: Selective Binding followed by Structural Re-arrangement

Argonaute (Ago) proteins and microRNAs (miRNAs) are central components in RNA interference, which is a key cellular mechanism for sequence-specific gene silencing. Despite intensive studies, molecular mechanisms of how Ago recognizes miRNA remain largely elusive. In this study, we propose a two-step mechanism for this molecular recognition: selective binding followed by structural re-arrangement. Our model is based on the results of a combination of Markov State Models (MSMs), large-scale protein-RNA docking, and molecular dynamics (MD) simulations. Using MSMs, we identify an open state of apo human Ago-2 in fast equilibrium with partially open and closed states. Conformations in this open state are distinguished by their largely exposed binding grooves that can geometrically accommodate miRNA as indicated in our protein-RNA docking studies. miRNA may then selectively bind to these open conformations. Upon the initial binding, the complex may perform further structural re-arrangement as shown in our MD simulations and eventually reach the stable binary complex structure. Our results provide novel insights in Ago-miRNA recognition mechanisms and our methodology holds great potential to be widely applied in the studies of other important molecular recognition systems.Author Summary: In RNA interference, Argonaute proteins and microRNAs together form the functional core that regulates the gene expression with high sequence specificity. Elucidating the detailed mechanism of molecular recognition between Argonaute proteins and microRNAs is thus important not only for the fundamental understanding of RNA interference, but also for the further development of microRNA-based therapeutic application. In this work, we propose a two-step model to understand the mechanism of microRNA loading into human Argonaute-2: selective binding followed by structural re-arrangement. Our model is based on the results from a combined approach of molecular dynamics simulations, Markov State Models and protein-RNA docking. In particular, we identify a metastable open state of apo hAgo2 in rapid equilibrium with other states. Some of conformations in this open state have largely exposed RNA binding groove that can accommodate microRNA. We further show that the initial Argonaute-microRNA binding complex undergoes structural re-arrangement to reach stable binary crystal structure. These results provide novel insights into the underlying mechanism of Argonaute-microRNA recognition. In addition, our method is readily applicable to the investigation of other complex molecular recognition events such as protein-protein interactions and protein-ligand binding.