Conformational Dynamics of thiM Riboswitch To Understand the Gene Regulation Mechanism Using Markov State Modeling and the Residual Fluctuation Network Approach

Abstract

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.

In silico investigation of riboswitches in fungi: structural and dynamical insights into TPP riboswitches in Aspergillus oryzae

“…We selected the three-dimensional structure of TPP riboswitch from Escherichia coli (PDB ID: 2GDI) [ 11 ] as a reference for constructing the Aspergillus oryzae TPP riboswitches models. We chose the template based on the crystal resolution (2.05 Å) and the availability of literature data regarding its structure and dynamics, which allows a reference parameter for simulation results [ 5 , 21 , 39 ]. The E. coli TPP riboswitch (TPPsw EC ) comprises the aptamer domain bound to one thiamine pyrophosphate molecule and three magnesium ions.…”

“…The P1 helix connects the aptamer domain to the expression platform, while P2 and P3 interact mainly with the pyrimidine ring and stems P4 and P5 with the pyrophosphate group of TPP ( Figure 1(b) ). Depending on the organism, divergences among species are found mainly in the P3 region, varying in sequence, extension, and base-pairing content [ 5 , 21 ]. In eukaryotic organisms, the P3 region is commonly more extensive than in prokaryotes.…”

“…In eukaryotic organisms, the P3 region is commonly more extensive than in prokaryotes. Besides, bacteria and archaea commonly show a ramification of the P3 stem, known as P3a, not found yet in eukaryotes [ 21 ].

Figure 1.

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“…Several studies have investigated the biotechnological and therapeutic potential of bacterial TPP riboswitches [ 14 , 21 , 22 ]. However, fungal TPP riboswitches are still poorly explored in terms of dynamics and ligand binding behaviour.…”

Riboswitch Folds to Holo-Form Like Structure Even in the Absence of Cognate Ligand at High Mg2+ Concentration

“…Riboswitches are important drug targets since they are involved in the critical functional role of gene regulation in bacteria and also absent in the human genome. 8,9 Experiments 10-22 and simulations [23][24][25][26][27][28][29][30][31] probing the folding of ADs in the presence of Mg 2+ and their cognate ligands provide evidence that the ADs populate multiple intermediates in their folding pathways. Functional studies on TPP, 32 Adenine, 33 Mg 2+ , 34 c-di-GMP, 35 SAM-I 36 and FMN 37,38 riboswitches further demonstrate that higher concentrations of cognate ligands are required than their intrinsic affinity to effectively regulate gene expression, indicating that riboswitch functioning is under kinetic control rather than thermodynamic control.…”