The Effect Of Mutations On The Interactions Of Four Proteins With Ligands Studied By Molecular Dynamics Simulations

Proteins carry out numberous roles and are involved in virtually all life processes in biological organisms.When crucial residues are mutated,they will cause a series of structural and functional changes of protein.The molecular simulations of proteins have already become a very important and active research field.At present,the site-directed mutagenesis has been used to establish molecular recognition and the reduced affinity caused by site-directed mutagenesis is important for understanding the mechanism of interaction.Combing with structural data,the mutations introduced by computational method are more inclined to analyze thermodynamic and kinetic parameters.In our thesis,molecular docking and molecular dynamics?MD?were used to theoretically study the interactions between receptor and ligand and investigate the influences of mutated residue on the structure and function in detail.The main results are summarized as follows: 1.The influence of residue in the position of 116 on the inhibitory potency of TH588 for MTH1MutT homolog 1?MTH1?is a Nudix pyrophosphatase and it's found in all classes of organisms.It hydrolyzes the oxidized purine deoxyribonucleotides and their ribonucleotide analogs,into monophosphate equivalents.The incorporation of the oxidized nucleotide into DNA causes mutations and cell death,which highlights that MTH1 is a new therapeutic target in cancer.TH588 is the first-in-class inhibitor that binds in the MTH1 active site,thus abrogating MTH1's activity.The study of TH588 against MTH1 reported that TH588 is 20-fold higher potency toward human MTH1 compared to mouse MTH1.The sequence alignment and structural comparison show that the residues forming the active site of the human MTH1 and mouse MTH1 are identical.The residue in position 116,neighboring the inhibitor binding site,is leucine in the mouse MTH1 and methionine in human MTH1.The MD simulations were used to study the different inhibitory potency of TH588 against human MTH1 and mouse MTH1 and to identify how residue in position 116 of MTH1 has an important contribution to TH588 affinity.The Leu116 disrupts the interactions between Asn33 and TH588,thus induces the conformational changes of Asn33 as well as TH588,which is different from that of Met116.The altered interactions between TH588 and mouse MTH1 change the flexibility of TH588,which could induce the remarkable conformational fluctuation of mouse MTH1.The more open binding site may explain the lower inhibitor potency of TH588 against mouse MTH1 than human MTH1.The effect of different residue Leu or Met in position 116 on the binding affinity of TH588 for MTH1 may be expected to contribute to the further rational design of more potent inhibitors.2.Molecular basis of the recognition of FMN by a HAD phosphatase TON₀338The HAD?haloalkaloic acid dehalogenase?superfamily,which is regarded as one of largest enzyme family,acts on a variety ofphosphorylated substrates.TON₀338,one of the HAD phosphatases in Thermococcusonnurineus NA1,is recently solved in CHES?N-cyclohexyl-2-aminoethanesulfonic acid?-bound complex.The residues Trp58 and Trp61 interact with ligand by forming a tryptophan sandwich structure during the simulation.The tested phosphatase activity of TON 0338 on representative metabolic phoshpocompounds shows that flavin mono-nucleotide?FMN?is the most preferred substrate among them.And the activity is largely reduced by the double-tryptophan mutation.MD simulations and the Molecular Mechanics/ Generalized Born Surface Area?MM/GBSA?free energy calculations were used to explore the effects of mutations on the changes in both structural flexibility and conformational dynamics of TON₀338 with FMN.The non-polar energy plays an indispensable role in the binding process of TON₀338 and FMN.The tryptophan sandwich structure provides a primary function to anchor FMN and keeps FMN well bound to TON₀338.The double-tryptophan mutation has influences on the secondary structures of TON₀338 and changes the conformation of cap domain,which would lead to reduced activity of W58A/W61A-FMN binding.The important insights of the structure-function relationships of TON₀338 protein could contribute to further understanding about the HAD phosphatases.3.Molecular dynamics simulation studies of influence of Tyr422 Ala mutation on TEAD4-TAZ and TEAD4-YAP complexesTranscriptional enhancer activation domain?TEAD?is the downstream transcriptional factor of the Hippo pathway.The TEAD proteins are crucial for cell development and also play a role in cancers.The transcription co-activators YAP?Yes-associated protein?and its paralog TAZ?transcription co-activator with PDZ-binding motif?,which are the key effectors of Hippo pathway,bind to TEAD to promote transcription of genes in cell proliferation and anti-apoptosis.The residue Tyr422 of mouse TEAD4 forms a hydrogen bond with Ser79 of YAP and Ser51 of TAZ,which is crucial and conserved.Point mutation of the Tyr422 residue of TEAD4 would disrupt the relevant hydrogen bond and even abolish the interaction.MD simulations were used to explore the effects of mutation Tyr422 Ala on the structural flexibility and conformational dynamics of mouse TEAD4 with the transcription co-activators YAP and TAZ.The non-polar interactions play an indispensable role in the binding process of TEAD4 and YAP/TAZ.The helices ?1 and ?2 of YAP/TAZ provide a primary function to anchor YAP/TAZ well bound to TEAD4.The binding conformation of TAZ/YAP distorted by decreased non-polar interaction and the lost hydrogen bonds would lead to reduced interaction activity.The present study would provide important insights into the structure-function relationships of TEAD protein and give a new explanation for the affinity of YAP/TAZ with TEAD.4.Molecular dynamics simulations studies and free energy analysis on inhibitors of MDM2-p53 interactionIn tumors,p53 protein is a potent inducer of cell cycle arrest,DNA repair,cellular senescence,innate immunity and apoptosis.The E3 ubiquitin ligase MDM2 is one of the principal p53 modulators,binding directly to the p53 transactivation domain,and inhibits p53-dependent transcription.Thus,reactivation of p53 by the inhibition of its binding to MDM2 is regarded as an effective and confirmed approach in cancer therapy.Recently,two peptide inhibitors pDI?LTFEHYWAQLTS?and pMI?TSFAEYWNLLSP?was identified using phage display.Using pDI and pMI for comparison,a quadruple mutant peptide?pDIQ,ETFEHWWSQLLS?was reported as the most potent inhibitor against MDM2.MD simulations were performed on three inhibitors-MDM2 complexes to investigate the stability and structural transitions.Molecular Mechanics/ Generalized Born Surface Area?MM/GBSA?approach was introduced to analyze the interactions between inhibitors and MDM2.The results show that binding energy of inhibitor pDIQ to MDM2 is significantly stronger than that of pMI and pDI to MDM2.The side chains of residues have more contribution than backbone of residues in energy decomposition.The residue Trp/Tyr22? is very important to the interaction between the inhibitors and MDM2.The three-dimensional structures at different times indicate that the mobility of Tyr100 influences on the binding of inhibitors to MDM2,and its change has important role in conformations of inhibitors and MDM2.The mutation of amino acids reveals the effect of mutated residue on the structure and binding activity of four proteins,which may provide important reference information for structure-based drug design and cancer therapy.