Molecular Dynamics Study Of Tryptophan And Its Derivatives To Inhibit β-amyloid Aggregation

Purpose:Alzheimer’s disease（AD）is one of many neurodegenerative diseases that affects the most people.The neurotoxicity caused by the accumulation ofβ-amyloid（Aβ）in the brain is confirmed to cause AD Key factors.Although researchers have long been committed to the study of Aβaggregation and its inhibition,effective therapeutic drugs have not been developed so far.There is an urgent need to find treatment methods that are non-toxic and side-effects and are not limited to the blood-brain barrier.Exercise can promote fat mobilization and induce the human body’s essential amino acid Tryptophan（Trp）to efficiently cross the blood-brain barrier.The amino acid level also strengthens the synthesis of the human body’s important neurotransmitter Serotonin（Serotonin,Ser）and the hormone Melatonin（Melatonin,Mel）.Experimental studies have found that tryptophan can inhibit the aggregation process of Aβ,destroy mature Aβfibers,and reduce the neurotoxicity of Aβfibers,and serotonin and melatonin have also been shown to inhibit the fibrosis process of Aβ,but the specific mechanism by which these molecules exert their inhibitory effects is still unclear.This study uses molecular dynamics simulations,replica exchange molecular dynamics simulations and other methods to explore the molecular mechanism of tryptophan and its metabolites（serotonin and melatonin）to inhibit/destroy Aβoligomers and Aβfibrils,which can provides an explanation perspective on the influence of exercise on the Aβfibrosis process and a certain theoretical support for elucidating the influence of exercise on the pathological process of AD,and also provides a structural basis and theoretical reference for the design of drug replacement therapy such as exercise intervention.Methods:In order to investigate the detailed mechanism of Trp and metabolites（Ser and Mel）affecting the Aβaggregation process,this study used all-atom replica exchange molecular dynamics simulation to explore the inhibitory mechanism of Trp,Ser and Mel on Aβoligomer aggregation.Moreover,this study using all-atom molecular dynamics simulation to study the mechanism of these small molecules dissociating Aβfibrils.All the all-atom molecular dynamics simulations in this study were performed using GROMACS software.GAFF force field was used to process the relevant parameters of Trp and Ser and Mel.The formal molecular dynamics simulation used AMBER99SB-ILDN force field.In addition,the data processing and analysis involved in this research are also carried out using GROMACS.At the same time,self-compiled scripts and programs and some third-party programs are used as supplementary tools for data analysis.Results:Elucidating the molecular mechanism of tryptophan and metabolites affecting the Aβaggregation process will help provide an explanation for the delay of the pathological process of AD by exercise.This study comprehensively used molecular dynamics and replica exchange molecular dynamics simulation methods to explore the detailed mechanism of tryptophan and metabolites that inhibit Aβaggregation,which is difficult to characterize in existing experiments.The main results of the research are as follows:（1）The addition of Trp effectively inhibits the formation ofβ-sheet structure in the secondary structure,especially in the N-terminal region 2AEF4,the central hydrophobic region 17LVF19 and the C-terminal region 34LMVGGVV40.At the same time,Trp also promotes the corresponding regions of the generation of coil and helix structure.Trp can weaken the interaction between the amino acid pairs in the Aβ₄₂dimer,and the weakening of the inter-chain interaction is more significant,with regard to the solution accessible surface area,inter-chain contact area,inter-chain contact number and inter-chain binding free energy yielded the same result.This study identified the main binding sites of Trp in Aβ₄₂dimers,including F4,Y10,F19,F20,I31,I32 and 34LMVGGV39.The results of studies on the interaction between Trp and Aβ₄₂fibrils show that the addition of Trp significantly reduces the structural stability of the N-terminal of Aβ₄₂fibrils and destroys theβ-sheet structure of the N-terminal of Aβ42 fibrils.The RMSF calculation results show that Trp is enhanced the flexibility of the N-terminal region.Trp can destroy the stability of peptide chains,reduce main chain hydrogen bonds,destroy the hydrophobic interaction of the local hydrophobic core HC1,and weaken the stability of K28 and A42 to form intra-chain and inter-chain salt bridges.The main binding sites of Trp and Aβ₄₂fibrils include F4,H6,Y10,H13,H14 and L34.（2）Ser can affect the secondary structure of Aβ₄₂dimer and inhibit the formation of beta structure in the 26SNKGAII32 region.By analyzing the results of residue-residue interaction,solution accessibility surface area,inter-chain contact area,contact number,and binding free energy,it can be seen that the addition of Ser significantly affects the overall and local inter-chain interaction of Aβ₄₂dimer.This study identified Ser and Aβ₄₂dimer binding to 4FRH6,Y10,13HHQKLVFF20,K28,I31,I32 and 34LMV36.In the analysis of the interaction between Ser and Aβ42fibrils,it is found that Ser can weaken the structural stability of Aβ42 fibrils,and has a more obvious effect on the N-terminal and C-terminal regions.In addition,the addition of Ser also largely destroyed the intra-chain and inter-chain salt bridges between K28-A42.Combining the calculation results of the number of contacts,the number of hydrogen bonds and theπ-πstacking form,it can be seen that Ser can weaken the hydrophobic interaction in HC1,destroy the N-terminalβ-sheet structure,and weaken the structural stability of the fibril.（3）The results of Cα-RMSD of Aβ₄₂fibrils show that Ser can increase Cα-RMSD stronger than Ser_pro.The calculation results of the gyration radius show that the protonated Ser_procan increase the gyration radius of the fibril to a certain extent.The results of the study on the influence of Ser/Ser_proon the number of contacts and binding energy between chains show that both Ser and Ser_procan reduce the number of contacts and weaken the binding energy between chains,and Ser_prohas a relatively strong effect.The analysis results of amino acids interaction show that although Ser/Ser_procan destroy the hydrophobic interaction between amino acids in HC1 and the K28-A42 salt bridge interaction,the destruction effect of Ser is stronger than Ser_pro.This study identified that aromatic interactions mainly drive the binding between Ser and binding sites F4,H6,Y10,H13,Q15 and L34,while aromatic interactions,hydrophobic interactions and electrostatic interactions together drive Ser_probinding to the binding sites D1,F4,R5,H6,D7,Y10,H13,Q15,F20,E22,D23and L34.However,although Ser_prohas a wider binding site and forms aπ-πstacking with more aromatic amino acids,the binding strength between Ser and the binding site is stronger and theπ-πstacking formed with the N-terminal of the fibril is more abundant,which enables Ser to destroy the stable structure of Aβ₄₂fibrils to a greater extent and depolymerize the formed Aβ₄₂fibrils.（4）The binding sites of Mel on Aβ₄₂dimer are almost all over the entire sequence.Mel weakens the possibility of beta structure formation in the N-terminal region and the central hydrophobic region,and inhibits further fibrillation of Aβ₄₂oligomers.Mel can reduce the mutual contact between the amino acid pairs in the chain and the amino acid pairs in the chain of the Aβ₄₂dimer,especially the influence on the chain interaction is more significant.The calculation results of the accessible surface area of the dimer solution,the contact area and number between the two peptide chains,and the binding free energy also show that Mel can weaken the interchain interaction between adjacent peptide chains.The results of the study on the interaction between Mel and Aβ₄₂fibrils show that Mel can increase the Cα-RMSD of Aβ42 fibrils,reduce theβ-sheet structure content of fibrils,and weaken the interchain stability of Aβ₄₂fibrils.At the same time,it reduces the contact of amino acids in the hydrophobic core and destroys the inter-chain and intra-chain salt bridges between K28 and A42.In addition,this study also identified Mel can be widely bound to the outer and inner surfaces of Aβ₄₂fibrils.（5）Trp and Trp＿Ser both increase the Cα-RMSD of Aβ₄₂fibrils,but although Trp＿Ser increases the Cα-RMSD of the Aβ₄₂fibril structure at the N-terminal and C-terminals,Trp can still increase the Cα-RMSD of the N-terminal and destroys the overall stability of Aβ₄₂fibrils to a greater extent.The results of RMSF,main chain hydrogen bond and Rg also showed that Trp changed the overall protein configuration from a compact structure to a loose structure to a greater extent.However,Trp＿Ser weakens the interaction between chains stronger than Trp.The analysis of the secondary structure shows that both Trp and Trp＿Ser can significantly reduce the probability of the formation ofβ-sheet at the N-terminal of the fibril.The analysis results of the stability of the hydrophobic core showed that Trp and Trp＿Ser destroyed the stable structures of the N-terminal and C-terminal of the fibril more obviously,and Trp＿Ser destroyed the K28-A42 salt bridge to a greater extent.This study also identified that in the Aβ₄₂＿protofibril+Trp and Aβ₄₂＿protofibril+Trp＿Ser systems,the binding sites of Trp and Trp＿Ser are very similar,almost all located at the N-terminal of the fibril.Conclusions:（1）Trp can inhibit the formation of ordered structures such as Aβ₄₂dimerβ-sheet,so that the protein conformation maintains a more disordered secondary structure under the inhibitory effect of Trp.Trp weakens the interaction between dimer chains and destroys the structural basis of Aβ₄₂fibrillation,thereby inhibiting the process of Aβ₄₂abnormal fibrillation.The binding of Trp to the N-terminal of Aβ₄₂dimer is mainly throughπ-πstacking and hydrogen bond interaction,while the binding to the C-terminal is mainly through hydrophobic interaction.At the same time,the addition of Trp significantly reduced the structural stability of Aβ₄₂fibrils,and the most significant impact occurred in the N-terminal region of Aβ₄₂.Trp formed more hydrogen bonds and abundantπ-πaccumulation with the N-terminal.It shows that Trp can damage Aβ₄₂fibrils at the N-terminal of Aβ₄₂,and then depolymerize the formed fibrils and inhibit the further aggregation of fibrils.（2）Ser effectively inhibited the formation of beta structure in the secondary structure of Aβ₄₂dimer in the 26SNKGAII32 region.Moreover,Ser greatly weakens the stable structure formed by the inter-chain interaction,thereby preventing the formation of stable aggregates.Electrostatic interaction,hydrophobic interaction and benzene ring interaction are jointly driven,and the hydrogen bond formed with the N-terminal charged amino acid are the main driving force to make Ser more bound to the N-terminal.In addition,Ser can weaken the structural stability of the N-terminal and C-terminal regions of Aβ₄₂fibrils,and destroy the N-terminalβ-sheet structure.This study found that Ser can rely on hydrogen bonding andπ-πstacking to combine with the N-terminus of Aβ₄₂fibrils,making the N-terminus a key area for depolymerization of Aβ₄₂fibrils.（3）Ser can weaken the stability of Aβ₄₂fibrils and promote the depolymerization of the stable fibrils that have been formed.Although protonated Ser_procan loosen the protein structure to a certain extent,it has less influence on the overall structure of the protein than Ser.The weaken effect of Ser_proon the inter-chain stability of fibrils is relatively stronger than Ser,indicating that Ser_promainly depolymerizes the formed Aβ42 fibrils by weakening the inter-chain stability.On the basis of weakening the inter-chain interaction,Ser can also destroy theβ-sheet structure of the N-terminal of Aβ₄₂fibrils by reducing the probability of formation of the N-terminalβ-sheet,thereby depolymerizing the Aβ₄₂fibrils.In addition,the binding of Ser to the binding site mainly relies on aromatic interactions,while the binding of Ser_proto the binding site is mainly driven by aromatic interactions,hydrophobic interactions and electrostatic interactions,which promotes the ability of Ser to be more effective than Ser_proto depolymerize the formed Aβ₄₂fibrils.（4）Mel can inhibit the further fibrillation of Aβ₄₂oligomers by weakening the formation probability of beta structure in the N-terminal region and the central hydrophobic region,and weakening the interaction between peptide chains.Mel can bind to almost the entire sequence of Aβ₄₂dimers throughπ-πstacking and hydrophobic interactions.The hydrophobic interaction is an important driving force for the binding of Mel to the C-terminal.At the same time,Mel can greatly reduce the structural stability of the N-terminal and C-terminal regions of Aβ₄₂fibrils.The analysis of the binding mode shows that the main driving force driving Mel to combine with the N-terminal and C-terminal isπ-πstacking.The role and hydrophobic interaction,and under the combined influence of these effects,Mel can largely destroy theβ-sheet structure of the N-terminal and C-terminal of the fibril.（5）Trp and Trp＿Ser destroy the stable structure of fibrils by weakening the overall stability of Aβ₄₂fibrils and the interaction between chains.Both Trp and Trp＿Ser can obviously destroy the stableβ-sheet structure at the N-terminal of the fibril,thereby depolymerizing Aβ₄₂fibril,and the depolymerization effect of Trp＿Ser is stronger.Due to the influence on water transport interaction and salt bridge interaction,Trp and Trp＿Ser have more obvious damage to the stable structure of the N-terminal and C-terminal of the fibril,respectively.The binding sites of Trp and Trp＿Ser are almost the same at the N-terminal of the fibril.Among them,the hydrogen bond interactions with N-terminal charged amino acids and N-terminal aromatic amino acids andπ-πstacking together drive the combination of small molecules and fibrils in the two systems.