Molecular Basis Of ABA-regulated Receptor Protein Activation And Targeted Molecular Design

Over the past half-century,droughts have reduced crop yields by 10%bringing the pressure to modern agricultural production,which is one of the most common and consequential natural disasters.Therefore,how to improve the drought tolerance of crops is a major scientific problem to ensure food security in the process of agricultural modernization in China.Abscisic acid(ABA)can effectively alleviate this problem and improve the stress tolerance of plants.A team got ABA receptor,PYR/PYL/RCARs(Pyrabactin Resistance 1/PYR-like Proteins/Regulatory Components of ABA Receptor)based on small molecule probe.The PYLs binding with ABA inhibits the activity of PP2C(Protein Phosphatase 2C).Additionally,SnRK2s(SNF1-related protein kinase 2)is released to active ABA signaling.Although chemical molecule probes play an important role in the study of the regulatory mechanism,the PYLs chemical regulators are rarely reported.The main reasons are as following:1.The redundancy effect in function of PYLs protein family members is notable,and the drought-resistance target is still unclear;2.PYLs can be divided into dimers and monomers,but the difference in ABA recognition between them is not clear.3.The mechanism of ABA-regulated PYLs conformational changes remains unclear.In conclusion,the molecular basis of PYLs remains to be further studied,and these studies are undoubtedly of great guiding significance for the rational design of PYLs small molecule regulators.This thesis mainly revolves around the above issues.Firstly,the molecular physiological mechanism of abscisic acid receptors has already been studied by the method of bioinfomatics.PYLs genes of several species were collected and classified.On this basis,we analyzed and compared the gene distribution,gene structure and gene expression of this protein family.At the same time,the evolutionary process of proteins was further studied based on phylogenetic tree.Subsequently,the family proteins were further categorized according to Motif.Finally,positive selection sites and functional divergence sites were calculated according to the classification.Meanwhile,the mutation and the corresponding binding free energy difference based on the crystal structure of PYL1 in arabidopsis thaliana was calculated,and the function of the corresponding sites on PYLs was clarified.Secondly,it is a very complicated task to study the conformational changes of ABA-regulated receptor PYLs protein.Therefore,the molecular dymanic methods were summarized and a comprehensive web server for exploring ligand-driven internal protein dynamics via molecular dynamics was built.Based on the CMD(Conventional Molecular Dynamics),SMD(Steered Molecular Dynamics)and NMA(Normal Mode Analysis)method and Bio3d,MDTraj tools,a fast and efficient LARMD program was developed.It provides standard protocols to prepare structures,build force field libraries for molecules,detect tunnel,run unbiased and biased molecular dynamics simulations,calculate energy and analyze trajectories.At the same time,LARMD web server(http://chemyang.ccnu.edu.cn/ccb/server/LARMD)was built up based on a powerful web platform(LAMP).The result files generated by the online server can be downloaded and viewed by users.The corresponding vector image files can be downloaded in PDF format.This powerful server can meet the needs of most non-professional researchers.It not only can be used to confirm the binding mode of small molecules and proteins,but also can simulate the small molecules binding and unbinding process in proteins.More importantly,the server provides a variety of conformational analysis methods,which can help us capture conformational changes more quickly and efficiently.Then,the molecular activative mechanism of PYLs was studied based on LARMD server.We used PYL2 as a template for molecular simulation.Based on the calculation results of Int_mod module,a long time conventional molecular dynamic was further carried out.And the molecular mechanism of ABA regulating receptor activation was studied by free energy calculation,umbrella sampling,the potential of mean force and so on.Our calculation results were consistent with previous hypothesis.The perception tunnels of ABA in monomers and dimers were different.However,the binding free energy of ABA with these two types of proteins were similar without any energy barrier.The low binding capacity of ABA in dimers was caused by thermodynamic penalties introduced by dimer depolymerization.At the same time,we developed an automatic calculating process of the potential of mean force,AUTO_PMF,which can be applied to other systems.The important residues predicted also lay a solid foundation for future work.Finally,screening the chemical molecule probes based on the molecular mechanism of PYLs activation.A novel automatic virtual screening software(AUTO CDVS)was designed based on consistency docking and consistency scoring.It was applied to design the chemical regulators of PYLs.We used this software to screen Specs and ChemDiv databases based on complex structure of PYR1-HAB1 and obtained the seed compounds 04554115 and 01444403.After optimization of 04554115,a lead compound with higher activty and good binding ability to PYR1 was obtained(Y18244).