Dynamics-based Recognition Mechanism of dsRBD-dsRNA interaction

Abstract

The broad range of cellular activities – ranging from cell-growth, development, to death – involves interaction between double-stranded RNAs (dsRNAs) and dsRNA-binding domains (dsRBDs). The dsRBDs are known to interact with A-form helical dsRNAs via minor-major-minor grooves spanning a length of about 12 base-pairs. A handful of dsRBDs in the cell are exposed to a large number of dsRNAs with an assortment of topologies. The variation in topologies is generated as a result of defects in the A-form helical structure. These defects result due to the frequent presence of mismatches and bulges in the RNA secondary structure. dsRBDs are known to target such topologically different dsRNAs with similar binding affinities. They are present as modular units in proteins like TRBP (TAR RNA-binding protein), ADAR (Adenosine deaminase acting on RNA), and Staufen, are involved in RNAi, RNA editing, RNA transport, respectively. Recent reports have shown that dsRBDs slides along the length of the dsRNAs having different secondary structures in an ATP-independent manner. These observations lead to the broader question that is “how do dsRBDs target a versatile range of dsRNA topologies?”. We hypothesized that conformational dynamics in the dsRBDs might play a role in the above-mentioned dsRBD-dsRNA interactions. We have employed two dsRBDs (from two different species – Homo sapiens and Drosophila melanogaster) as model systems (TRBP2-dsRBD1 and dADAR-dsRBD1, respectively) that have similar secondary and tertiary fold but different primary sequence. TRBP has three dsRBD domains which it uses to target dsRNA (with dsRBD1 and dsRBD2) and bind to Dicer (with dsRBD3) so that Dicer, an endonuclease, can act on target dsRNA sites. On the other hand, ADAR contains two N-terminal dsRBDs that allows binding to dsRNAs and a deaminase domain at C-terminus that targets dsRNAs to carry out A-to-I editing post-transcriptionally. We first performed a systematic study of dsRBD dynamics in absence and presence of substrate dsRNAs of multiple topologies using NMR spectroscopy. NMR is a unique technique that allows to access atomic level information on motions occurring at timescale ranging from ps to s or more without the need of any chemical modification. While determination of ps-ns timescale dynamics showed the presence of differential dynamics in the two dsRBDs, a similar pattern of motions occurring at μs-ms timescale with faster μs timescale motions present in dsRNA binding region was observed. Additionally, an allosteric effect of dynamics was observed in the two dsRBDs as presence of motions at μs-ms timescale was detected all along the backbone of the protein instead of localized in the RNA-binding regions. We next explored the binding of the two dsRBDs with multiple dsRNAs using Isothermal Titration Calorimetry (ITC) and NMR-based titrations. These data showed that the interaction between the model dsRBDs and the dsRNAs occurred at the μs timescale and depends mostly on the dsRNA shape. It also showed that any enthalpic change in the binding reaction due to RNA-shape is compensated by the corresponding change in entropy to keep the free energy of binding unperturbed. Further characterization of motions in dsRNA-bound state revealed that high-frequency motions (at μs timescale) present in free dsRBDs are transferred to the neighboring residues when bound to the substrate dsRNAs. Thus we conclude that the μs-ms timescale dynamics present in dsRBDs are responsible for adaptation required to interact with topologically different dsRNAs.