Theoretical Study On The Interaction Of Cytochrome P450 5A1 And 11B1 With Ligands

Cytochrome P450 are widely present in a variety of organisms and are involved in a variety of important physiological processes such as detoxification,defence and biosynthesis of endogenous compounds（e.g.steroid hormones）as well as the metabolism of exogenous substances.The development of many diseases is associated with the metabolism of P450 enzymes and it is important to understand the interaction of P450 enzymes with ligands.The use of experimental methods alone to study protein function has major limitations and has been difficult to meet the needs of people,which are filled by computer technology-based molecular simulation methods.In this thesis,molecular dynamics simulations were used in combination with other analytical methods to systematically investigate the structure and properties of two P450 enzymes（CYP5A1 and CYP11B1）.The main contents are summarized as follows:1.Study on the Interaction between CYP5A1 and Inhibitors Using Molecular Dynamics SimulationsCytochrome P450 5A1（CYP5A1）is involved in the synthesis of thromboxane A₂,a potent mediator of platelet aggregation,vasoconstriction and bronchoconstriction.And CYP5A1 is an important target for the treatment of cardiovascular diseases.However,the lack of crystal structure has hindered the in-depth understanding of the structural characteristics of enzymes to some extent,and the molecular mechanisms of CYP5A1 and inhibitors are still unclear.This work examined the interaction of CYP5A1 with three inhibitors（ozagrel,pyragrel and furegrelate）by means of homology modelling,molecular docking and molecular dynamics（MD）simulations.The results showed that three inhibitors were stabilized in the binding pocket of CYP5A1 in a similar binding mode.Binding free energy calculation results show that hydrophobic interaction is a key factor in the binding of CYP5A1 to inhibitors.The hydrogen bond interation between inhibitors to CYP5A1 also play a role in their binding.Residues L125,I340 and I345 play an important role in the binding of CYP5A1 and the three inhibitors.They form a’clamp’structure that holds inhibitors in the binding pocket.In addition,we found that inhibitor pyragrel has a stronger binding affinity compared to the other two inhibitors due to more branched chains and more contact with the protein.This work will help us to better understand the structure-function relationship of CYP5A1 and may provide theoretical clues for the development of related inhibitors.2.The Molecular Mechanism of CYP11B1 Mutants Affecting Substrate BindingCytochrome P450 11B1（CYP11B1）belongs to the cytochrome P450 family 11subfamily and is involved in the synthesis of sterol.CYP11B1 mutations will decrease the level of cortisol and increase the level of steroid precursors,which ultimately cause congenital adrenocortical hyperplasia（CAH）.Therefore,it is important to understand residue mutations for the structure and function of CYP11B1.The literatures report that the A306V and L299P mutations in CYP11B1 cause an almost complete loss of the enzyme activity.11-deoxycorticosterone（DOC）is a typical substrate of CYP11B1.In this part of the work,we examined the interaction between CYP11B1 and DOC using molecular docking,molecular dynamics simulations,binding free energy calculation,free energy landscape and tunnel analysis using DOC as a molecular probe to clarify the details of their interaction and elucidate the reasons for the reduced enzymatic activity of CYP11B1 due to the A306V and L299P mutations.9 key residues（F130,A313,G314,T318,V378,G379,L382,F487,and I488）constitute a hydrophobic pocket that stabilizes DOC in the active site through hydrophobic interactions.And F381 forms an important hydrogen bond with DOC anchoring DOC in the favorable position for catalysis.Mutation studies showed that the mutation of A306 to V306indirectly destroy the hydrophobic cavity and thus affects substrate binding.While the mutation of L299 to P299 causes a local conformational change in CYP11B1,which ultimately affects substrate binding.In conclusion,the results show that A306V and L299P mutations indirectly change the binding mode of DOC and decrease the affinity between ligand and CYP11B1,which are unfavorable for the occurrence of reactions.The results of the study can help us understand how CYP11B1 metabolizes DOC and explain at the atomic level why mutations lead to the reduction of CYP11B1 activity.