Binding-induced functional-domain motions in the Argonaute characterized by adaptive advanced sampling

Argonaute proteins in combination with short microRNA (miRNAs) can target mRNA molecules for translation inhibition or degradation and play a key role in many regulatory processes. The miRNAs act as guide RNAs that associate with Argonaute and the complementary mRNA target region. The complex formation results in activation of Argonaute and specific cleavage of the target mRNA. Both the binding and activation processes involve essential domain rearrangements of functional importance. For the Thermus Thermophilus Argonaute (TtAgo) system guide-bound (binary) and guide/target-bound (ternary) complexes are known but how the binding of guide and target mediate domain movements is still not understood. We have studied the Argonaute domain motion in apo and guide/target bound states using Molecular Dynamics simulations and a Hamiltonian replica exchange (H-REMD) method that employs a specific biasing potential to accelerate domain motions. The H-REMD technique indicates sampling of a much broader distribution of domain arrangements both in the apo as well as binary and ternary complexes compared to regular MD simulations. In the apo state domain arrangements corresponding to more compact (closed) states are mainly sampled which undergo an opening upon guide and guide/target binding. Whereas only limited overlap in domain geometry between apo and bound states was found, a larger similarity in the domain distribution is observed for the simulations of binary and ternary complexes. Comparative simulations on ternary complexes with 15 or 16 base pairs (bp) formed between guide and target strands (instead of 14) resulted in dissociation of the 3’-guide strand from the PAZ domain and domain rearrangement. This agrees with the experimental observation that guide-target pairing beyond 14 bps is required for activation and gives a mechanistic explanation for the experimentally observed activation process.Author summary: Post-transcriptional gene silencing is an important process to regulate protein synthesis in eukaryotes and prokaryotes. The Argonaute proteins as part of the RNA-induced-silencing-complex (RISC) form a central element of the process by silencing of a target messenger RNA (mRNA) via degradation or repression of translation. The Argonaute protein binds initially a short RNA that acts as a guide to promote binding of a complementary target mRNA. The complex formation can lead to activation of Argonaute and specific cleavage of the target mRNA. The whole process involves domain rearrangements that are not fully understood. We applied an advanced Molecular Dynamics sampling technique to specifically accelerate domain motions of the Thermus Thermophilus Argonaute (TtAgo) system in apo, guide bound and guide/target bound states. The simulations indicate only limited overlap of domain arrangements in apo and bound states and identified domain opening motions necessary for guide and target binding. The study also offers an explanation why a minimum of 15 or 16 base pairs between guide and target strands are necessary for Argonaute activation.