Metal Ion Mediated Riboswitch Folding

Ribonucleic acid (RNA) exhibits a series of biological activities such as protein synthesis, catalytic activity, gene regulation, and gene editing through the formation of specific three-dimensional structures. Riboswitches are regulatory genetic elements located in the 5’-untranslated region of several prokaryotic mRNAs. Typically, riboswitches comprise an aptamer domain and an expression platform. The aptamer domain secondary structure is highly conserved and undergoes a conformational change in response to specific ligand binding. These conformational changes affect the expression platform and can alter gene expression through different mechanisms, including transcriptional attenuation, sequestration of the ribosomal binding site, or ribozyme-mediated degradation. Purine riboswitches are highly conserved across different organisms and have been extensively studied. This class of riboswitches includes those that specifically recognize and bind to guanine or adenine. The ability to differentiate between guanine and adenine in each riboswitch is determined by a single nucleotide (C or U) located within the binding pocket of the aptamer domain. One notable example of the purine riboswitch class is the adenine riboswitch (add riboswitch), which functions as an ’on’ switch for the expression of adenine deaminase. When cellular adenine concentrations reach a certain threshold, the binding of adenine to the aptamer induces a conformational change that activates translation of the gene encoding adenine deaminase by exposing the Shine-Dalgarno sequence.

Metal ions are important in the folding and stabilization of complex RNA structures. Due to the polyanionic character of the RNA phosphodiester backbone, it inevitably brings negative charges in close proximity to each other. This results in highly unfavorable electrostatic interactions that are anisotropically distributed in the folded form of the RNA. Therefore, divalent metal ions in general and Mg$^{2+}$ with its favorable charge/size ratio in particular play an important role in RNA folding. Ions bind to RNA through two binding modes – inner-sphere (IS) (direct ion-RNA contact) and outer-sphere (OS) (water-mediated ion-RNA contact) coordination. While many studies of add riboswitch have been performed, the role of ions on add riboswitch is still not fully understood at the atomistic level of detail. In my research work, I am investigating the role of metal ion in the stabilization of the adenine riboswitch (add-riboswitch) using unbiased all-atom molecular dynamics simulations with GROMACS software.

Polymer physics studies the mechanical properties and kinetics of polymers. Polymer collapse refers to the phenomenon where a polymer chain, initially in a stretched or extended conformation, undergoes a transition to a more compact or collapsed state. This transition can occur due to numerous factors, such as changes in temperature, solvent conditions, or interactions with other molecules. Understanding polymer collapse is crucial in comprehending the behavior of polymers in different environments and their potential applications. The study of polymer collapse provides insights into the selfassembly and folding of proteins, which are essential for understanding biological processes. The better the solvent the greater the 'swelling' of the molecule. Conversely, the poorer the solvent the greater the ‘collapse’ of molecule. At equilibrium, the average size of isolated polymer molecules in solution is a strong function of the quality of the solvent, and varies from expanded conformations in good solvents, to random walk conformations in theta solvents, and collapsed conformations in poor solvents.

5-Lipoxygenase (5-LOX) is an enzyme involved in the biosynthesis of leukotrienes, potent inflammatory mediators that play a critical role in various diseases, such as asthma and cardiovascular disease. Despite its importance, the mechanism of oxygen insertion in 5-LOX into its substrate (arachidonic acid, ACD) still needs to be better understood. In this study, we used network analysis and molecular dynamics simulations to investigate the oxygen migration pathway of 5-LOX in its closed and open conformations. Our results show that the closed conformation of 5-LOX is more stable than the open conformation and that the addition of substrate induces a compactness in the enzyme. To examine the oxygen migration pathway, we clustered the oxygen trajectories and found 11 distinct oxygen pockets in the substrate-bound 5-LOX. We characterized the oxygen pockets in the active site of 5-LOX and found that the enzyme can make four different regio-selective products. Since hydrogen is abstracted from C7 of ACD, both CS and C9 are potential positions of oxygen attack. Based on direction of attack, 5S, 95, and 9R products are possible. These apart, we also find evidence for a 158 product. Our study provides novel insights into the conformational dynamics and oxygen insertion mechanism of 5-LOX and may have important implications for developing new drugs targeting this enzyme.