Inherent conformational plasticity in dsRBDs enables interaction with topologically distinct RNAs

Differential conformational dynamics in two type-A RNA-binding domains drive the double-stranded RNA recognition and binding

“…Furthermore, there were two intriguing observations in the 1 H- 15 N HSQC-based titrations. First, the amide signals were getting broadened at as low as 0.1 RNA equivalents, suggesting the slow-to-intermediate timescale of binding as has also been observed previously by our and other research groups in TRBP2 dsRBD1, Staufen dsRBD3, hDus2 dsRBD, MLE dsRBD2, DBR4 dsRBD1, PKR dsRBD, and Dicer dsRBD (Ankush Jagtap et al, 2019; Bou-Nader et al, 2019; Chiliveri et al, 2017; Paithankar et al, 2022; Ramos et al, 2000; Ucci et al, 2007; Wostenberg et al, 2012; Yadav et al, 2019). Second, not only the reported RNA-binding residues but the entire backbone was undergoing line broadening, suggesting the presence of RNA-induced motions in the entire backbone.…”

“…Briefly, wt miR-16 (22–23 bp) has a bulge (unpaired uridine) and an internal loop (A:A mismatch), miR-16-1-M has only A:A mismatch, (ii) miR-16-1-B has only U-bulge, and (iii) miR-16-1-D has neither the bulge nor internal loop forming a perfect duplex (Figure 1 of (Paithankar et al, 2022)). It has been established that the TRBP2-dsRBD1 is able to recognize a set of topologically different dsRNA structures owing to its high conformational plasticity (Paithankar et al, 2022). In the current study, we have characterized the interaction of TRBP2-dsRBD2 with the same set of topologically different dsRNAs (wt miR-16-1 and mutants) used for TRBP2-dsRBD1 through 1 H- 15 N HSQC-based NMR titrations shown in Figure S7.…”

“…Briefly, the respective guide and passenger RNA listed in Supplementary Table S1 were mixed together in a 1:1 ratio, followed by denaturation at 90°C for 10 min and then cooling at 4°C for 30 min. The annealing of all four RNAs (miR-16-1-A, miR-16-1-M, miR-16-1-B, and miR-16-1-D) (Paithankar et al, 2022) and D12 RNA was confirmed by 1 H NMR by observing the imino proton signals (Figure S1). The annealed samples were maintained in buffer D for all data measurements.…”

“…The number of residues with kex > 5,000 Hz and > 10% pB (red and green, medium and big spheres) (Figure 4C) differed vastly between the two domains. Only 3 residues with such conformational exchange in dsRBD2 were identified: R200 (loop 3), L212 and R224 (α2); whereas in dsRBD1, the number is 15, distributed along the entire backbone of the protein (Figure 6 of (Paithankar et al, 2022)), similar to PKR-dsRBD1, where 75% of the residues from dsRBM1 showed Rex > 1 s -1 (Nanduri et al, 2000). Interestingly, all three conserved RNA-binding regions in dsRBD1: α1 (L35, Q36), β1-β2 loop (E54), and α2 (N61) were brimming with a kex > 50,000 Hz.…”

“…TRBP and its homologs across different species such as Loquacious (Loqs in D. melanogaster ), R2D2 ( D. melanogaster ), DRB1-3,5 ( A. thaliana ), RNAi defective 4 (RDE-4 in C. elegans ), Xlrbpa ( X. laevis ) (Eckmann and Jantsch, 1997), and the PKR activator (PACT) protein (mammals) (Peters et al, 2001) have conserved arrangement of three consecutive dsRBDs; of which, the two N-terminal domains (type A) are known to bind dsRNAs, whereas the third domain is known for protein-protein interactions. We have recently established the role of TRBP2-dsRBD1 dynamics in dsRBD-dsRNA interactions and proposed that dsRBD1 adopts a conformationally dynamic structure to recognize a set of topologically different dsRNA structures (Paithankar et al, 2022). The µs timescale motions were found to be present all along the dsRBD1 backbone with higher frequency motions ( k ex > 50 kHz) in the RNA-binding sites.…”