Research On Structural Modeling And Interaction Of Two Important Proteins

In the activities of life,proteins are the main participants.They function by interacting with other proteins or small molecules,while these interactions are based on their tertiary structures(3D-structures).Therefore,determining the 3D-structures of proteins is the basis of understanding its interaction mechanism with other proteins or small molecules,and it is also the basis for designing protein inhibitors,drugs and so on.Due to the fact that many3D-structures of proteins have not yet been determined by experimental methods,it is necessary to model the 3D-structure based on the theoretical modeling to study their interactions and design inhibitors,drugs and so on.The work in this research aims at two proteins with important application value,constructing their theoretical 3D-structures and studying their interactions with small molecules.It includes the following three aspects:1.We modeled the 3D-structure of marine low temperature alkaline protease MP,studied its interaction with small molecule inhibitors,and screened three reversible inhibitors.Protease MP is a low temperature alkaline protease from marine bacteria,which has good prospects in application.However,the instability of liquid enzyme has become a bottleneck restricting its large-scale application.It is of great significance to screen a reversible inhibitor to break through the bottleneck of the application.Firstly,we modeled the 3D-structure of MP by using the method of structural modeling,which was later determined by X-ray diffraction experiments with the RMSD between our modeled structure being 0.11?,and the differences of these two structures merely in the region of non-active center.Secondly,based on previous experimental information,molecular docking technology was used to develop screening strategies and identify pharmacophore models based on the structure of the modeled structure,and screened the molecules in the compound database ZINC.Finally,three molecules were proved to have reversible inhibition on MP.2.The open state 3D-structure of the voltage-gated sodium channel Na_v1.5 was modeled.Its interaction with drugs was studied and the binding sites were given.The voltage-gated sodium channel Na_v1.5 is encoded by the gene of SCN5A and it is specifically expressed in the cardiac tissue.When its function is abnormal,it can cause the channelopathy.Due to the lack of the atomic structure of Na_v1.5,the research and development of antiarrhythmic drugs and their mechanisms were seriously hindered.In this study,the open structure of the Na_v1.5 pore structure was constructed by ROSETTA-membrane homology modeling method.And then analyzed interaction mechanisms of Na_v1.5 and four open states local anesthetic drugs(Flecainide,Pilsicainide,Cocaine and Ranolazine),which shows that the residues F526(F1760 Nav1.5?),Y533(Y1767 Na_v1.5?)and other sites play important roles in the combination of local anesthetic drugs and the open-state Na_v1.5.This research will provide a structural basis for channel drug development and drug action mechanism study.3.Based on the co-evolution analysis of protein residues,we constructed the3D-structure of the voltage gated sodium channel pore in closed state and studied its interactions with drugs.In the process of protein evolution,in order to maintain the protein functional or structural stability,residue interactions keep in a"co-evolution"mode,i.e.when a residue was mutated,its interacting residues were also mutated correspondingly to maintain their interactions.Based on this,we firstly modeled the Na_v1.5 pore domains in closed state through the evolutionary information between residues of Na_v1.5,and then using the method of molecular dynamics simulation and MM-PBSA,we analyzed the interaction mechanism between Na_v1.5 and three closed-state drugs(Pilsicainide,Bisphenol A and Mexiletine)and shows that the residue PHE512(F1760 Na_v1.5?)plays a key role in the association of three closed-state local anesthetics with the closed-state Na_v1.5.This result is consistent with the previous experimental results and it confirms the accuracy of our structure from another aspect.The results of this study can help to elucidate the action mechanism of closed-state channel drugs.