Fig 1.
Visualization of the seven metastable macrostates obtained from MSM of apo hAgo2.
(A) Distribution of the PAZ-PIWI loops center-of-mass (c.o.m.) distances for each macrostate. A large distance implies an open conformation. The equilibrium population of each macrostate is presented. (B) Projections of the open and partially open states onto PAZ-PIWI loops c.o.m. distance and the major PIWI loop angle (see S3 Fig for the angle definition). The green cross corresponds to the binary partially open crystal structure (missing residues modeled). (C) Representative structures of closed, partially open and open states. Enlarged view of the inter-domain region between PAZ (red) and PIWI (green) of each structure is presented in the inset panel (see representative structure for each macrostate in S4 Fig).
Fig 2.
Projection of hAgo2-miRNA docking models built from the selected structures of open microstates.
Red dots mark the successful docking models and black dots mark the unsuccessful ones. A successful docking model is a hAgo2-miRNA docking pose where at least two native contacts are preserved at each miRNA terminus.
Fig 3.
Binary docking models undergo substantial structural re-arrangement towards binary crystal during MD simulations.
(A) Projections of MD trajectories of three representative successful docking models. The rationale behind the choice of representative docked conformations is simply to select conformations that contain different fractions of native contacts (i.e. the lowest: 27.1%; median: 35.4%, and highest: 50.0%). (B) Upper panel: interface-RMSD (iRMSD) of models against binary crystal structure. Lower panel: fraction of native contacts (fnat) between hAgo2 and miRNA of the models. (C) Structural comparison between the binary crystal structure (left) and a representative model from MD (right) with hAgo2 colored in grey and miRNA in red.
Fig 4.
The proposed two-step model of miRNA loading into hAgo2: Selective binding followed by structural re-arrangement (highlighted by the cyan arrow).
The induced fit mechanism (marked by the upper right grey arrow) and the conformational selection mechanism (marked by the lower left grey arrow) are also presented to compare with the two-step model. Average transition times between the closed states, the open state and the partially open states are computed from ten independent 10-ms synthetic trajectories generated by sampling the transition probability matrix of the 480-microstate MSM (see S10 Fig for additional details). The detailed transition pathways from the closed states to the open state can be found in S11 Fig.
Fig 5.
Mutations in the binding pocket for miRNA(5') show different effects on hAgo2-miRNA binding.
(A) miRNA(5’)-hAgo2 distance is defined as the minimum distance between U1 of miRNA and its binding pocket in hAgo2 (K533, Q545, K566, K570 and R812). The increase of the distance is an indicator of the weaker hAgo2-miRNA interactions. (B) Time traces of miRNA(5’)-hAgo2 distance in wild type (WT) hAgo2 (blue), the Y529A mutant (green) and the Y529E mutant (red). Error bars were computed from five independent MD simulations.
Fig 6.
Mutations in PIWI loops destabilize the closed conformation and accelerate the closed-to-open transitions.
Three mutants were generated: D823A (green), E821A-D823A-E826A (red) and ∆602–605∆819–833 (a deletion mutant where both PIWI loops are truncated, cyan). MD simulations of WT hAgo2 (blue) and the mutants were initiated from three conformations: (A) a closed conformation, (B) a partially open conformation extracted from the binary hAgo2-miRNA crystal structure (PDB ID: 4F3T) and (C) an open conformation. Time traces of the c.o.m. distance between the PAZ domain and PIWI loops of the WT hAgo2 and the three mutants are displayed. Error bars were computed from five independent MD simulations.