Research
Research
Introduction:
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 struc- tures. The metal ions play a crucial role in stabilizing the functionally active structure of the RNA by neutralizing its negatively charged phosphate backbone. Experiments can provide the structure of ion-bound small fragments of RNA molecules and provide insights into multiple aspects of RNA folding in the presence of metal ions. However, inferring the populations of different thermody- namic states and their detailed microscopic structure is challenging in experiments. In addition, the mechanism through which the metal ions interact with the RNA especially the formation of tertiary interactions in the presence of monovalent alkali metals, divalent alkaline earth metal, and divalent transition metal ions remains elusive. In my research work, I am investigating the mechanism of metal-ion condensation over RNA and its implication in RNA using the techniques of all-atom molecular dynamics simulations with GROMACS-2018.6 software and PLUMED-2.5.1 software combinedly.
Free Energy Landscape of GAAA Tetraloop Receptor Complex in Presence of Metal Ions
Loop-receptor interactions are common in tertiary structural elements of RNA. The specific interactions between GNRA tetraloop (where N is any nucleotide and R is guanine or adenine) and its receptor is the classic example of loop-receptor complex, and it is stabilized by metal ions. To investigate the role of metal ions on the stability of loop-receptor complex we have selected GAAA tetraloop and its receptor, an 11 nucleotides long RNA segment found in group 1, group II introns, and RNase P as a model system and studied folding thermodynamics of GAAA tetraloop-receptor complex in the presence of Na+ and Mg2+.
The fluorescence resonance energy transfer (FRET) experiment provides evidence that a high concentration of monovalent ion ([Na+]) and divalent ion ([Mg2+) facilitate the docking of GAAA tetraloop to its receptor. Experiments further confirm that high [Na+] screens electrostatic repulsion among the phosphate atoms and RNA charges are strongly neutralized due to an increase in [Mg2+] which makes diffusive ions ([Na+]) distribute more uniformly (large entropy). This enhanced entropy brings down the free energy of the GAAA tetraloop-receptor system. However, experiments are unable to provide the atomistic description of metal ion binding to the tetraloop-receptor region and its role in the docking-undocking process. To probe the docking-undocking mechanism as a function of ion concentration we performed all-atom molecular dynamics simulations in the presence of metal ions. The free energy barrier separating the docked state from the undocked state is high and difficult to sample the conformational space using conventional all-atom simulations. To overcome the limitation of conventional all-atom simulations, I used well-tempered meta-dynamics (WT-MetaD) technique, in which a systematic adaptive bias is added to the basins visited by the system to explore the free energy surface at a faster rate.
We have used GROMACS-2018.6 and PLUMED-2.5.1 to perform WT-MetaD simulations in the presence of Na+ and a mixture of Na+ and Mg2+. We have constructed the free energy surface of the GAAA tetraloop-receptor complex as a function of two collective variables - the distance between two pairs of residues connecting tetraloop and receptor, and, eRMSD with respect to the set of three bases of the complex's crystal structure reported in protein data bank (PDB) (PDB ID: 1GID). At a smaller distance between the loop and receptor, and a smaller eRMSD value, the docked state is thermodynamically stable. The stability of the docked state is attributed to the presence of monovalent ion, Na+ in the cavity of GAAA tetraloop, which screened out the electrostatic repulsion among the phosphate backbone and assists the formation of three base pairs between tetraloop and its receptor. The larger values of distance and eRMSD signify the undocked state, which is devoid of Na+ ion in the cavity of the loop-receptor complex. On going from docked to undocked state, there are two intermediate states which have the same number of base pairs as the undocked state, but unlike the undocked state, the intermediates are occupied by the Na+ ions. We observe different intermediates between docked and undocked states in which tetraloop and tetraloop-receptor are present in close proximity without forming hydrogen bonding. A cavity is formed in between GAAA tetraloop and tetraloop receptor arms and the number of Mg2+ ions are increasing in this cavity during the transition from undocked to docked state. A total of five residues (two from GAAA tetraloop arm and three from tetraloop receptor arm) were involved in the formation of hy- drogen bonds with water molecules of first solvation shells of Mg2+ ions (outer sphere co-ordination) which assist the docking of GAAA tetraloop receptor complex. Diffused Na+ ions were replaced by tightly bound Mg2+ ions in that cavity with the increase in [Mg2+] in the docked state. As a result, the lifetime of the docked state is increasing with higher [Mg2+]. The findings of our simulations are amenable to experiments.
Local RNA Structure, Ion Charge Density and Ion Hydration Shell Determine the Mechanism of Specific Ion Condensation on RNA
NiCo riboswitch which can bind extremely toxic metals like Ni2+ and Co2+ along with other alkali and alkaline earth metal ions, is a perfect system to probe the binding mechanism of various metal ions with RNA residues. In this work, we took 31 nt long P2 arm of NiCo riboswitch system to study the interaction of different metal-ion (Na+, K+, Mg2+, Ca2+, Ni2+, Mn2+, Co2+, and Zn2+) RNA interaction using all-atom molecular dynamics simulation. We observe that the mode of binding of metal ion does not solely depend on the charge density of the ion (ρc). In general higher the ρc, the higher will be the binding lifetime. Monovalent ions follow this trend. How- ever, divalent ions which have smaller ρc have a greater binding lifetime with RNA except Ni2+. Although Ni2+ has the highest ρc, it has a moderately high binding lifetime than that of Zn2+, Co2+, and Mg2+. The ion binding lifetime through inner shell coordination (direct binding of a metal ion with RNA) is higher than that of outer shell coordination (water-mediated binding with RNA). Alkali metals having the smaller ρc, have the affinity to bind via inner shell coordination. Among alkaline earth metals, Ca2+ has a higher ion binding lifetime and lower barrier height for one water exchange from first to second solvation shell. For transition metal ions, we observe that the ion binding lifetime of Mn2+ is higher than other ions, and also water exchange barrier of Mn2+ is lower. So, Mn2+ and Ca2+ have smaller ρc which assists those ions to lose water molecules from the first solvation shell and bind with RNA via inner shell co-ordination. The high barrier height of the water exchange process for Mg2+, Zn2+, and Co2+ ions suggest that they can only interact with RNA through water-mediated outer shell co-ordination. The switching of outer to inner coordination while interacting with P sites of RNA increases with lowering the energy barrier of the water exchange process. We observe one-way switching of interaction from outer to inner shell coordination mode for Ni2+ and Mn2+. But once they are in outer shell coordination mode, they can not switch back to inner shell co-ordination mode. But for alkali metal (Na+ and K+) and Ca2+, reversible switching can be observed.
Transition Metal Cation Helps to Fold Four Way Junction in NiCo Riboswitch by Outer-Shell coordination in Low Ion Concentration Regime
We have taken full NiCo riboswitch to study the role of metal ions (Mg2+, Ni2+, Co2+, Zn2+) in tertiary contacts formation between two arms (P2 and P4) of NiCo riboswitch. For each system, two crucial hydrogen bonds are formed between four residues of P2 and P4 arms which stabilize the interaction between the two arms. As a result, two helices of P2 and P4 come in close proximity to form a cavity where one metal ion can sit and interact with residues via outer shell coordination. After around 1 μs simulation time, among the four metal-ion water systems, we observe Ni2+ and Zn2+ can present significantly for a longer time in the cavity with the formation of water-mediated hydrogen bonds than Mg2+ and Co2+. This is because the radius of hydrated Ni2+ and Zn2+ ion are smaller than that of Mg2+ and Co2+. In the future, I will probe the reason for the specific interaction of metal ions with different arms of NiCo riboswitch.
How Does an RNA Fragment Sense a Metal Ion?
RNA structural stability and function depend upon interactions with intracellular cations, primarily with divalent cations like Mg$^{2+}$ and Ca$^{2+}$. Non-coding RNA molecules possess a unique capability to sense certain cations and carry out specific biological functions. However, the microscopic insight of specific ion sensing by RNA remains elusive. Ions bind to RNA through two binding modes \textendash \ inner-sphere (direct ion-RNA contact) and outer-sphere (water-mediated ion-RNA contact) coordination. Due to the high energy barrier of transition between the two binding modes, it is challenging to sample both modes using conventional unbiased molecular dynamics simulations. To overcome the sampling problem, we performed well-tempered metadynamics simulations and probed how this tetraloop specifically senses divalent metal ions through inner and outer shell coordination. We observed both modes of RNA-cation interactions and investigated the role of the hydration layer around ions in the binding process. Our simulation results revealed that the free energy surface (FES) of this small RNA fragment is multidimensional. Our finding suggested that due to lesser charge density of Ca$^{2+}$ compared to Mg$^{2+}$, Ca$^{2+}$ can undergo rapid outer-to-inner shell transition than that of Mg$^{2+}$. The structural flexibility of this small tetraloop plays a critical role in giving stability to structures with various inner-shell contacts. With increase RMSD from 0.1 nm to 0.4 nm, the RNA structure is more flexible to easily form more than one inner-shell contacts. RMSD of 0.68 gives fully extended chain like structure
Less flexible structures can sample mostly one inner-shell contact. With increased flexibility, base oxygens and phosphate oxygens can form simultaneous inner-shell contacts. For that reason, the unfolded structure of the tetraloop in presence of both Mg$^{2+}$ ion and Ca$^{2+}$ ion can form simultaneously greater number of inner shell contacts more easily than the folded and semi-folded structures. Maximum four inner shell interactions can be formed with Mg$^{2+}$ and RNA oxygen atoms, whereas Ca$^{2+}$ can bind up to five RNA heavy atoms (oxygen and nitrogen) with inner shell coordination.