A Computational Study Of Riboswitches To Identify Small Molecules Of Ligands

The riboswitches are a class of RNA structural elements found in non-coding regionsand that can combine with small molecular metabolites to regulate the transcription and translation of downstream target genes through conformational changes.The riboswitches are highly conserved,with high specificity and selectivity of ligand recognition,which make them act as a target of the new class of nucleic acid drugs and novel antibiotic.The purine riboswitches specifically recognize the purine and the purine analogues.Because of the simple structure and the crystal structure of the purine riboswitches,it has become one of the most widely studied model systems.In spite of the purine riboswitches have been studied extensively in biochemistry,genetic structure and other experiments,and some theoretical studies,the little detailed information of binding between the purine riboswitches and the ligands has been revealed and need to do more work.Molecular dynamics(MD)simulations can analyze the characteristics of the interaction between the purine riboswitches and the ligands in atomic level,and connect the microstructure information of interaction with the experimental macroscopic characteristics.MD simulations also help us understand the molecular recognition mechanisms and can provide theoretical support to develop the new drug targets and new antibiotic targets of RNA.In this paper,MD simulations and thermodynamic integration methods were used to study the essence of specific recognition between the purine riboswitches and the ligand analogues,as well as the mechanism of the conformational change and the high selectivity of the ligands.The all-atom MD simulations of the systems were carried out by using AMBER force field and TIP3 P water molecular model.We are focused on the interaction of four ligands(GUA,6GU,2BP and XAN)with two different types of the purine riboswitches(GR,the guanine riboswitch and GRA,its variant C74U)and the conformational changes of the riboswitches.Firstly,the thermodynamic integration was used to calculate the relative binding free energy of the guanine riboswitch(GR)with four ligands and GR complexes with its variant C74U(GRA)complexes.Secondly,we analyzed the hydrogen bonds between ligand molecules and two types of riboswitches,the recognition of the key residues to ligands and the comparison between 6GU/2BP complex and GUA complex.Finally,the mutation of C74 into U74,a key residue of the purine riboswitches,and the conformational changes of ligand binding were analyzed.The main results can be seen in the following paragraphs.1.The relative binding free energy is calculated by the thermodynamic integration method.The relative binding free energies of GUA –GR complex relative to the other three complexes were calculated by thermodynamic integration.We also predicted the relative binding free energies of the GR complexes relative to the GRA complexes.The results show that the rank of the predicted values is in agreement with the experimental data.What's more,the contributions of van der Waals interaction and polar interaction are different in the complexes of the same riboswitch with different ligands,as well as in the complexes of the different riboswitches with the same ligand.It also indicated that the polar interactions play an important role in the selectivity of the purine riboswitches.2.Identification and Interaction between ligands and the key nucleotides of the RNA.The analysis of hydrogen bonds and the free energy decomposition method,implies that there are six key residues with large contribution in the ligand recognition,they are A21,U22,U51,A52,C74 and U75.The nucleotides U51 and C74 are mainly electrostatic interactions by the hydrogen bonds.While the nucleotides A21,U22,A52,and U75 are the base stacking interactions,which is driven by van der Waals forces.3.Conformational changes in ligand bounding.Long time MD simulations(1?s)revealed that the conformations of GR are divided into two states: the closed state of the binding ligand and the open state of the unbound ligand.GR shows a modest folding in ligand binding.The conformational reorganization leads to a large conformational change when the ligand is removed.4.The influence of C74 mutation into U74.The results show that the high selectivity of GR mainly depends on the nucleotide 74.When C74 mutated to U74,the affinity of GUA was change largely.Compared to the other purine analogues,U74 and C74 recognize different ligands with different conformation and binding model.It is a guide to design new high selectivity ligands in the future.This thesis consists of five chapters.The first chapter is the introduction,which mainly introduces the relationship between the function and the structure,the classification and the control mechanism of the RNA,and the current international research progress and the future application prospect of the purine riboswitches.The second chapter mainly introduces the basic concept of MD simulation,including the basic theory of MD simulation,force field and its classification.Application of AMBER software and MD simulation in the study of the purine riboswitches are also introduced.In the third chapter,the research object and method are introduced.This part mainly introduces the preparation of the systems the MD simulation and the thermodynamic integration.The fourth chapter is the results and analysis.The relative binding free energies of GUA-GR relative to the other three complexes and GR complexes relative to GRA complexes were calculated by the thermodynamic integration.And the comparison between the predicted value and the experimental value was carried out.The hydrogen bonds between four ligands and the two kinds of riboswitches were calculated.The key nucleotides for ligand were identified by free energy decomposition method.The comparison of binding patterns between different complexes,the effect of C74 mutation on U74 and conformational changes in ligand bounding were also studied.The fifth chapter is the summary and prospect,summarizes the main conclusions and innovations of this paper.It is hoped that this study will be helpful to understand the recognition between the purine riboswitches and ligands,and provide theoretical support for the designing and developing the new drugs.