Fig 1.
(A) NMR structure of pre-miR20b RNA (pdb 2n7x). (B) NMR structure of the Rbfox•pre-miR20b complex (pdb 2n82). The residues at the binding interface are highlighted. The amino acids labelled with a red square correspond to the mutated residues in Rbfox* and in Rbfox* S151T. (C) Left: nucleotide sequence of the pre-miR20b and of the mutant pre-miR20b*. Right: amino acids sequence of the Rbfox, Rbfox* and Rbfox* S151T mutants. Highlighted in green and in orange are the amino acids corresponding to β strands and α helices, respectively. The mutated nucleotides and amino acids are underlined.
Table 1.
Simulations performed in this work.
Fig 2.
Fluctuations of the PAD angle in the simulations of free (top; see Table 1, sim. 1) and bound Rbfox protein (bottom; see Table 1, sim.
9–13). F indicates fluctuations; t short transitions and T long transitions. The structured regions of the protein are highlighted: the grey and yellow regions correspond to the β-strands and helices, respectively.
Fig 3.
(A) 2D representation of the free pre-miR20b stem-loop structure. (B) Time development of the εRMSD of the pre-miR20b loop (r-U27GGCAUG33) in the six χOL3 MD simulations (Table 1, sim. 2–7). C. RNA backbone dihedral angle histograms calculated over the aggregated simulations. The green dots indicate the values of the angles in the lowest energy structure of the NMR ensemble 2n7x from which the simulations were started.
Fig 4.
(A) Bird’s-eye view of the Rbfox•pre-miR20b complex. (B)-(H) Close-up views of the interactions observed at the binding interface during MD simulations. The H-bonds are indicated by dotted red lines. The depicted snapshots belong to the representative structures of the 20 clusters (“MD-adapted structure ensemble”), which have the highest agreement with NMR NOE data. (I) Scheme of the interactions. Circle and arrowheads depict interaction with RNA bases or phosphate groups, respectively.
Table 2.
Hydrogen bonds at the binding interface of the Rbfox•pre-miR20b complex observed during the MD simulations (Table 1, sim. 8–13).
The average donor-acceptor distances and angles are calculated over the entire simulation ensemble for the trajectory frames in which the individual H-bonds are observed. The interactions are further characterized by their occupancy in the individual simulation trajectories 8–13.
Table 3.
Percentage of intra- and intermolecular NOE violations observed in the course of the simulations of the protein-RNA complex.
Trajectory 8 (Table 1) has been obtained without applying the restraints in the initial stages of the simulation–see Methods.
Fig 5.
PAD and tag analyses for Rbfox* free (top) and bound to pre-miR20b* RNA (bottom). “F” indicates fluctuations; “t” short transitions and “T” long transitions. The secondary structure regions of the proteins are highlighted as in Fig 1.
Table 4.
Conformational entropy differences associated with Rbfox* protein for residues belonging to the β2β3 loop.
ΔS values are calculated as SRbfoxF—SRbfox*F (ΔSa) and SRbfox*F—SRbfox*C (ΔSb), over simulations 1 (Rbfox free), 14 (Rbfox* free), and 17 (Rbfox* bound) as listed in Table 1. The subscripts F and C refer to the free and bound proteins, respectively. These values are obtained with very approximate methods, and they should be taken only for qualitative comparisons.
Fig 6.
(A) Bird’s-eye view of the Rbfox*•pre-miR20b* complex. (B) and (C) Close-up views of the interactions established by the mutated residues with A30 and C33, respectively. (D) Scheme of the interactions between pre-miR20b* and Rbfox* observed in the MD simulations. Circle and arrowheads depict interaction with RNA bases or phosphate groups, respectively.
Table 5.
Hydrogen bonds at the binding interface of the Rbfox*•pre-miR20b* complex during simulations.
The average donor-acceptor distances and angles are calculated over the entire simulation ensemble for the trajectory frames in which the individual H-bonds are observed and the interactions are further characterized by their occupancy in the individual simulation trajectories. The trajectories are numbered as in Table 1.
Table 6.
Hydrogen bonds at the binding interfaces of the Rbfox•pre-miR20b* and Rbfox*•pre-miR20b complexes in MD simulations.
The average donor-acceptor distances and angles are calculated for the trajectory frames in which the individual H-bonds are observed and the interactions are further characterized by their occupancy.
Fig 7.
(A) Close up view on the interactions established by A30 with T151 and S155 and by U28 with S155 and F126. (B) Comparison between β2β3 loop and adjacent residues PAD values of the Rbfox* and S151T mutant in complex with pre-miR20b* RNA. The PAD values relate to the flexibility of the complex.