Thiamine pyrophosphate (TPP) riboswitch is a cis-regulatory element in the noncoding region of mRNA. The aptamer domain of TPP riboswitch detects the high abundance of coenzyme thiamine pyrophosphate (TPP) and modulates the gene expression for thiamine synthetic gene. The mechanistic understanding in recognition of TPP in aptamer domain and ligand-induced compactness for folding of expression platform are most important to designing novel modulators. To understand the dynamic behavior of TPP riboswitch upon TPP binding, molecular dynamics simulations were performed for 400 ns in both apo and TPP bound forms of thiM riboswitch from E. coli and analyzed in terms of eRMSD-based Markov state modeling and residual fluctuation network. Markov state models show good correlations in transition probability among metastable states from simulated trajectory and generated models. Structural compactness in TPP bound form is observed which is correlated with SAXS experiment. The importance of junction of P4 and P5 is evident during dynamics, which correlates with FRET analysis. The dynamic nature of two sensor forearms is due to the flexible P1 helix, which is its intrinsic property. The transient state in TPP-bound form was observed in the Markov state model, along with stable states. We believe that this transient state is responsible to assist the influx and outflux of ligand molecule by creating a solvent channel around the junction region of P4 and P5 and such a structure was anticipated in FRET analysis. The dynamic nature of riboswitch is dependent on the interaction between residues on distal loops L3 and L5/P3 and junction P4 and P5, J3/2 which stabilize the J2/4. It helps in the transfer of allosteric information between J2/4 and P3/L5 tertiary docking region through the active site residues. Understanding such information flow will benefit in highlighting crucial residues in highly dynamic and kinetic systems. Here, we report the residues and segments in riboswitch that play vital roles in providing stability and this can be exploited in designing inhibitors to regulate the functioning of riboswitches.

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Chikungunya virus is a growing human pathogen transmitted by mosquito bite. It causes fever, chills, nausea, vomiting, joint pain, headache, and swelling in the joints. Its replication and propagation depend on the protease activity of the Chikungunya virus-nsP2 protein, which cleaves the nsP1234 polyprotein replication complex into individual functional units. The N-terminal segment of papain is structurally identical with the Chikungunya virus-nsP2 protease. Hence, molecular dynamics simulations were performed to compare molecular mechanism of these proteases. The Chikungunya virus-snP2 protease shows more conformational changes and adopts an alternate conformation. However, N-terminal segment of these two proteases has identical active site scaffold with the conserved catalytic diad. Hence, some of the non-peptide inhibitors of papain were used for induced fit docking at the active site of the nsP2 to assess the binding mode. In addition, the peptides that connect different domains/protein in Chikungunya virus poly-protein were also subjected for docking. The overall results suggest that the active site scaffold is the same in both the proteases and a possibility exists to experimentally assess the efficacy of some of the papain inhibitors to inhibit the Chikungunya virus-nsP2.

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