Molecular Simulation Study On The Mechanism Of Misfolding And Aggregation Of Tau Protein

Tau is a microtubule-associated protein,and it can act as a core in the early stages of microtubule assembly after binding to tubulin,promoting the aggregation of other tubulins on this core to form microtubules and maintain the stability of its structure.Tau dysfunction is associated with many neurodegenerative diseases commonly known as tauopathies,including Alzheimer's disease(AD).Neurofibrillary tangles(NFTs)with ?-sheet rich structures formed by misfolding and aggregation of tau are the main pathological process of tauopathy.Therefore,investigating the mechanism of tau protein misfolding and aggregation is very important to understand the pathogenesis of tauopathy.In the present,the experimental methods have great limitations in the study of protein misfolding and aggregation,for example,the structure of misfolded proteins is difficult to be obtained by traditional experimental methods.Even if the static structure is obtained,it is difficult to provide the dynamic process of conformational transition.Therefore,so far,the pathogenesis of tauopathies is unclear.Compared with experimental methods,molecular dynamics(MD)simulation has unique advantage in studying the protein structural transformation.In this paper,combining molecular dynamics simulation and multiple trajectory analysis methods,we have investigated the conformational changes of tau protein during misfolding and aggregation and the inhibition mechanism of small molecule,mainly including the following four aspects:1)The dissociation mechanism of tau fibril boundary chains was investigated by steered molecular dynamics(SMD)simulation to reveal the misfolding mechanism of tau protein induced by template.The results show that the dissociation mechanisms of boundary chain in PHF and SF are different.In PHF,the environment of the chains on both sides of the pentamer is different,and the A chain is more easily dissociated.The dissociation path is divided into five steps.Firstly,the earliest dissociating region is ?2 and ?3,followed by the dissociation of peptides ?1,?4 and the beginning part of ?5.After that,the end part of ?5 and Loop?5-6 regions begin to dissociate.This is followed by the dissociation of ?6 and ?7.Finally,the ?8 dissociates.However,in SF,the environment of the chains on both sides of the fibril is similar,and the dissociation path of A and E chains are also similar.They are divided into five stages.It is simultaneously dissociated from ?1 and ?8 and ends at ?5.2)The molecular mechanism of spontaneous aggregation and template-induced conformational changes of key tau fragments was investigated by long-time molecular dynamics simulation.Our results indicate that PHF6 can spontaneously aggregate into heterogeneous oligomers enriched with ?-sheet structure including dimer,trimmer,and tetramer,and the peptide chains have a preference to adopt parallel ?-sheet.Under template induction,PHF6 monomer can be induced to form ?-sheet structure on either side of the template but in different ways.In detail,the ?-sheet structure is easier to be formed on the left side of the template but have a short ?-sheet structure.However,on the right side of the template,the monomer is induced by three steps to form an extended ?-sheet structure.Furthermore,MSM analysis shows that the formation of dimer mainly experiences three steps.Firstly,the separated monomers collide with each other at random orientations,and then a dimer with short ?-sheet structure at the N-terminal forms,finally the ?-sheet elongates to form an extended parallel ?-sheet dimer.3)The misfolding mechanism of the key segment R3 of tau protein was studied by combinding discrete molecular dynamics simulations and Markov state model.The results show that R3 monomer exists in disordered structures mainly,which is consistent with the experimental results.The MSM analysis identified multiple ?-sheet conformations of R3 monomer.The residues involved in ?-sheet structure formation are mainly located in three regions: PHF6 at the N-terminal,S324-N327 at the middle of R3,and K331-G334 at the C-terminal.In addition,path analysis to form ?-sheet by transition path theory(TPT)revealed that multiple paths could form ?-sheet structures from disordered state,and the timescales occur at millisecond level,indicating that a large number of structural rearrangements occur during the formation of ?-sheet structure.4)The molecular mechanism of inhibition of tau aggregation by small molecule inhibitor C30 was studied by all-atom molecular dynamiscs simulation combined with multiple analytical methods.The results show that C30 destroys the ordered structure of PHF6 oligomer,reduces the content of ?-sheet structure and transforms the ?-sheet into random coil structure.In addition,C30 has four possible binding sites on tau oligomer.Following a more in-depth analyses of each site,it is determined that the S1 site is the most possible binding site mainly located between the layers of L1 and L3.Energy analysis revealed that the hydrophobic interaction is the driving force for the binding of C30 to PHF6 oligomer.In addition,Y310 and V309 are key residues for C30 binding to oligomer.The fully understanding the inhibitory mechanism of 2,4-thiazolidinedione derivative on PHF6 oligomer and the identification of binding sites will help design and discover new AD inhibitors in the future.The above findings elucidate the mechanism of tau misfolding,aggregation,template-induced fiber elongation,and inhibition of tau aggregation by small molecule at the atomic level,which will help to understand the pathogenesis of AD and other tauoupathies and provide important theoretical guidance for the design of potential therapeutic drugs.