(A) dsRBD constructs [TRBP2-dsRBD1 (1–105 aa) and TRBP2-dsRBD2 (154–234 aa] of Human TRBP2 full-length protein used in this study. (B) Sequence alignment of the two constructs mentioned in (A). CS-Rosetta structures of (C) TRBP2-dsRBD1 (Paithankar et al., 2018), (D) TRBP2-dsRBD2, (E) an alignment of core residues of dsRBD1 and dsRBD2, (F) Model structure of 12 bp duplex D12 RNA.

(A) RMSF profile (by Cα) of core dsRBD1 (black) and dsRBD2 (red). The secondary structure for the two domains has been shown on the top, and three RNA-binding regions in dsRBD1 and dsRBD2 have been highlighted using vertical grey and yellow bars, respectively. (B) RMSD of profiles of dsRBD1 (black) and dsRBD2 (red) over 1 μs simulation time. RMSD values for data measured in triplicate have been separated by vertical dashed lines.

Spin relaxation parameters (A) R1, (B) R2, and (C) [1H]-15N nOe plotted against the residues for (left panel) apo TRBP2-dsRBD2 and (right panel) D12-bound TRBP2-dsRBD2. Experiments were recorded on a 600 MHz NMR spectrometer at 298 K. The secondary structure of TRBP2-dsRBD2 has been mentioned at the top, and the RNA-binding region of the protein has been marked in grey vertical columns. Average R1, R2, and [1H]-15N-nOe of the core residues (159-227 aa) at 600 MHz is depicted in the green bar, and at 800 MHz is depicted in the red bars.

(A) R1ρ data, (B) R2ρ data, recorded on apo 15N-TRBP2-dsRBD1, (C) R1ρ data, (B) R2ρ data, recorded on apo 15N-TRBP2-dsRBD2 using Heteronuclear Adiabatic Relaxation Dispersion (HARD) experiments on a 600 MHz NMR spectrometer at 298 K, plotted against residue numbers. An increase in the spin-lock field strength is achieved by an increase in the stretching factor of the adiabatic pulse used to create the spin lock, n (in HSn). The secondary structure has been depicted on the top, and three RNA-binding regions have been highlighted using vertical grey bars. Mapping of conformational exchange parameters (rate of exchange between the ground state and excited state (kex), and excited state population (pB)) obtained by fitting the above-described data to a two-state model on the CS-Rosetta structures of (E) apo-dsRBD1, and (F) apo-dsRBD2. Residues have been marked in different colors to highlight the distribution of kex values, and the diameters of the sphere indicate the extent of pB along the protein backbone. The RNA-binding residues have been depicted in stick mode.

Conformational exchange in (A) apo- and (B) D12-bound-TRBP2-dsRBD2. Top panel: Rate of exchange between the ground state and excited state (kex); Bottom panel: excited state population (pB) as obtained by the geometric approximation method, using the HARD experiment, plotted against residue numbers. Mapping of core kex, and pB, on the CS-Rosetta structure of apo TRBP2-dsRBD2, as extracted for (C) apo TRBP2-dsRBD2 and (D) D12-bound TRBP2-dsRBD2. Different colors highlight the distribution of kex values, and the sphere’s diameter indicates the extent of pB along the protein backbone. The RNA-binding residues have been depicted in stick mode.

ITC-based binding study of D12 duplex RNA with TBRP2-dsRBD1 and TRBP2dsRBD2. Top panel: the raw differential potential for each injection is plotted against the titration time for (A) TBRP2-dsRBD1 & (B) TBRP2-dsRBD2. Bottom panel: the integrated heat (enthalpy change) upon each injection (black dots) and the data fit for a single set of binding sites (red line) plotted against per mole of injectant for (C) TBRP2-dsRBD1 & (D) TBRP2-dsRBD2.

Conformational exchange perturbations in core TRBP2-dsRBD2 in the presence of D12 RNA. (A) Δkex (D12-bound – apo) TRBP2-dsRBD2 plotted against residue numbers. The secondary structure has been shown on the top, and three RNA-binding regions have been highlighted using vertical grey bars. Only residues having significant perturbation (Δkex > 10 kHz) have been plotted, where an increase is shown in red, and a decrease is shown in blue, (B) An increase in kex (red) and a decrease (blue) in the presence of D12 RNA indicated on the backbone of the CS-Rosetta structure of apo TRBP2-dsRBD2. The RNA-binding residues have been depicted in stick mode in the tertiary structure.

The model proposed for the two type-A dsRBDs in TRBP2 protein. dsRBD2 with rigid and conserved RNA binding regions is able to bind the RNA tightly, whereas dsRBD1 with high conformational exchange is able to recognize different RNA structures (often with bulges and internal loops). Following this, the two dsRBDs upon contacting the RNA undergoes enhanced conformational exchange at different extent. This enhanced conformational exchange coupled with differential binding affinity towards dsRNA might enable the tandem dsRBDs to move along the backbone of the RNA molecule, leading to ATP-independent diffusion.