Biology subjects

Paithankar, H.

Publications and source records attributed to Paithankar, H..

4 recordsLinked to original sources

bioRxiv2023

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

TAR RNA binding protein (TRBP) has emerged as a key player in the RNA interference (RNAi) pathway, wherein it binds to different pre-miRNAs and siRNAs, each varying in sequence and/or structure. We hypothesize that TRBP displays dynamic adaptability to accommodate heterogeneity in target RNA structures. Thus, it is crucial to ascertain the role of intrinsic and RNA-induced protein dynamics in RNA recognition and binding. We have previously elucidated the role of intrinsic and RNA-induced conformational exchange in the double-stranded RNA-binding domain 1 (dsRBD1) of TRBP in shape-dependent RNA recognition. The current study delves into the intrinsic and RNA-induced conformational dynamics of the TRBP-dsRBD2 and then compares it with the dsRBD1 study carried out previously. Remarkably, the two domains exhibit differential binding affinity to a 12 bp dsRNA owing to the presence of critical residues and structural plasticity. Further, we report that dsRBD2 depicts constrained conformational plasticity when compared to dsRBD1. Although, in the presence of RNA, dsRBD2 undergoes induced conformational exchange within the designated RNA-binding regions and other residues, the amplitude of the motions remains modest when compared to those observed in dsRBD1. We propose a dynamics-driven model of the two tandem domains of TRBP, substantiating their contributions to the versatility of dsRNA recognition and binding. Significance StatementExploring the intricacies of RNA-protein interactions by delving into dynamics-based measurements not only adds valuable insights into the mechanics of RNA-protein interactions but also underscores the significance of conformational dynamics in dictating the functional outcome in such tightly regulated biological processes. In this study, we measure intrinsic and RNA-induced conformational dynamics in the second dsRBD, i.e., TRBP-dsRBD2, and compare the same with that carried out in the first dsRBD (TRBP-dsRBD1) of TRBP protein, a key player of the RNAi pathway. The study unveils the differential conformational space accessible to the two domains of TRBP, even though they both adopt a canonical dsRBD fold, thereby affecting how they interact with target RNAs.

biophysics↗

bioRxiv2019

Role of protein dynamics in enthalpy-driven recognition of topologically distinct dsRNAs by dsRBDs

Taking leads from the fact that a handful of double-stranded RNA-binding domains (dsRBDs) interact with a massive number of topologically distinct double-stranded RNAs (dsRNAs) in crucial biological pathways, and to understand the adaptability required by dsRBDs to target the pool of dsRNA substrates, we employed two independent model dsRBDs in an ITC and NMR spectroscopy based study. Our previous study revealed the presence of microsecond timescale dynamics in RNA-binding regions in the two dsRBDs studied. In the current study, results from ITC-based titrations showed that the binding of dsRBD with topologically distinct dsRNAs is enthalpy-driven, with each dsRNA-dsRBD pair having distinct combination of enthalpy-entropy yielding a similar change in free energy upon RNA-binding. We also show that the each of the dsRNA, used in this study, binds to the dsRBD in a unique mode. Further, intrinsic conformational exchange present in the RNA-binding regions of the apo-dsRBD was shown to quench upon binding with a dsRNA, while conformational exchange got induced at the residues that are in close proximity to where exchange was present in the apo-protein. This apparent relay of conformational exchange from one site to the other site upon dsRNA-binding thus suggests the importance of intrinsic dynamics to adapt to target a variety of dsRNA-shapes. Statement of SignificanceThis study reports that the interaction between dsRBDs and dsRNAs is enthalpy-driven, is perturbed by subtle changes in dsRNA shapes, and exhibits a classic case of enthalpy-entropy compensation. Further, the intrinsic microsecond timescale conformational exchange present in the apo-dsRBD was observed to get quenched upon RNA-binding. An apparent relay of conformational exchange was also observed from quenched sites to spatially proximal sites upon RNA-binding, suggesting highly adaptive nature of the dsRBD, a critical feature required to target topologically distinct dsRNA.

biophysics↗

bioRxiv2019

Characterization of conformational dynamics at microsecond timescale in the RNA-binding regions of dsRNA-binding domains

Double-stranded RNA-binding domains (dsRBDs) are involved in a variety of biological functions via recognition and processing of dsRNAs. Though the primary substrate of the dsRBDs are dsRNAs with A-form helical geometry; they are known to interact with structurally diverse dsRNAs. Here, we have employed two model dsRBDs - TAR-RNA binding protein and Adenosine deaminase that acts on RNA - to understand the role of intrinsic protein dynamics in RNA binding. We have performed a detailed characterization of the residue-level dynamics by NMR spectroscopy for the two dsRBDs. While the dynamics profiles at the ps-ns timescale of the two dsRBDs were found to be different, a striking similarity was observed in the s-ms timescale dynamics for both the dsRBDs. Motions at fast s timescale (kex > 50000 s-1) were found to be present not only in the RNA-binding residues but also in some allosteric residues of the dsRBDs. We propose that this intrinsic s timescale dynamics observed independently in two distinct dsRBDs allows them to undergo conformational rearrangement that may aid dsRBDs to target substrate dsRNA from the pool of structurally different RNAs in cellular environment. Statement of SignificanceThis study reports for the first time the detailed characterization of microsecond timescale dynamics observed in RNA-binding regions of two distinct double-stranded RNA-binding domains (dsRBDs) using NMR relaxation dispersion experiments. dsRBDs have been known to target topologically distinct dsRNAs. However, the mechanistic details of the structural adaptation of proteins is not fully understood. We propose that the presence of such dynamics may have large-scale implications in understanding the RNA recognition mechanisms by the dsRBDs.

biophysics↗

bioRxiv2019

NMR-based analysis of nucleotide π-stacking in a crowded environment: Implications for prebiotic reactions

The inherent heterogeneity of the prebiotic milieu is often overlooked when studying nonenzymatic reactions. However, it is important to note that the prebiotic soup of a putative RNA World would have been replete with a plethora of molecules resulting from complex chemical syntheses, as well as exogeneous delivery. The presence of such background molecules could lead to pertinent phenomenon such as molecular crowding, which can potentially affect how a reaction would advent in a crowded milieu. In the current study, we have analyzed the effect of crowding on the stacking ability of the RNA monomers, using Nuclear Magnetic Resonance (NMR) spectroscopy. Our findings corroborate that the purine monomers possess better stacking efficiency than pyrimidine based monomers. Significantly, this competence is further enhanced in the presence of a crowding agent. Interestingly, this enhanced stacking could result in higher sequestration of the purine monomers, putting their ready availability for relevant nonenzymatic polymerization and replication reactions into question. Taken together, this study demonstrates the need for systematic biophysical characterization of molecular crowding in the context of prebiotically pertinent processes. Unravelling such phenomena is essential to gather a real understanding of how the transition from abiotic to biotic, would have happened during the origin of life.

biophysics↗