Theoretical Study On The Factors Of Proton Conduction Of BM2 Channel And The Interactions With The Channel Blocker

Highly contagious acute respiratory infection caused by influenza virus(referred to as influenza)is a serious threat to human life and health and socioeconomic development.Seasonal flu causes millions of severe cases and hundreds of thousands of deaths each year,according to the World Health Organization.Human influenza is mainly induced by influenza A and B viruses,which account for about 50% of all influenza virus infections.M2 channel,located on the envelope of influenza virus,can be activated and transfer protons in acidic environment,which plays an important role in the replication,budding and virus lifecycle.Blocking the proton conduction of M2 channel could prevent the virus from uncoating,then prevent the release of viral RNA into the cytoplasm and interrupting the early replication of the virus,which plays an important role in anti-influenza virus.Effective inhibitors targeting influenza B virus have been absent yet.Drug design of anti-influenza B virus is an urgent need to deal with the outbreak of influenza.Highly conserved M2 channel of influenza B virus(BM2)is regarded as an ideal anti-influenza target.The study on proton conduction of BM2 channel is significant to investigate the physiological mechanism of BM2 and provide the theoretical basis for the drug development of anti-influenza B virus.BM2 channel performs its physiological functions in the form of homotetramers.In 2009,through mutation and proton flow experiments,James J Chou et al.showed that mutations of the polar serine triplet located at the N-terminus of BM2 channel significantly reduced the proton conductivity of the channel.The NMR results from Mei Hong Group and fluorescence spectrum data from Gai Feng Group indicated that the conformation of BM2 and channel hydration related to proton conduction,may be affected by negatively charged membrane environment and membrane containing cholesterol.The above results revealed the key factors of proton conduction of BM2 channel,which played a positive role in understanding the mechanism of proton conduction of BM2 channel.However,the details of microscopic dynamic mechanism were still unclear.At present,to understand the microscopic mechanism of proton conduction of BM2 channel(including activating channel and transferring proton),we need to further research the key factors of proton conduction of BM2 channel and clarify the detailed mechanism,which is of great practical significance for experimental exploration and drug development against influenza B virus.With the development of science and technology,the study of the relationship between protein structure and function by biochemical experiments could not meet the increasing needs of researchers,which becomes a big challenge of studying protein function.Molecular simulations based on computer simulation technology emerges.In recent years,with the development of molecular simulation technology,the application of molecular simulations in complex biological systems such as proteins provides a new approach for the prediction of protein structure and function and the construction of protein models.At the same time,it provides a powerful supplement to the research fields that are difficult to reaseach by experimental methods,such as the dynamic information of protein-inhibitor interactions and the allosteric mechanism of channel proteins,and achieves the cross-link of multiple disciplines and fields.Nowadays,molecular simulation has been widely used in chemistry,physics,biology,medicine,materials and other research fields.With the help of computer technology,molecular simulation has become a powerful tool for studying the structure and function of proteins.In this paper,the structure and function of BM2 channel and the interactions of BM2 channel and the channel blocker were systematically and deeply studied by various molecular dynamics simulations.The main research contents of this paper are as follows:1.The effects of polar residues on BM2 channelIn order to clarify the mechanism of serine triad regulating proton conduction of BM2 channel,we performed molecular dynamics simulations and calculated the potential of mean force(PMF)to investigate serine triad regulating conformation,hydration and proton conduction of BM2 channel at the atomic level.Molecular dynamics simulations showed that serine triad could form stable hydrogen bond networks with His19 tetrad,which is in favor of the open states of the C-terminus of BM2 channel and allows more water molecules enter the channel.Thus,the hydration of BM2 channel increased more.We utilized Adaptive Streed Molecular Dynamics(ASMD)simulations to simulate hydrated proton diffusion and calculated PMF.The results show that the polar environment formed by serine triad is beneficial for proton diffusion of BM2 channel.This work elucidated the regulation mechanism of serine triad on the conformation,hydration and proton conduction of BM2 channel at the atomic level,which helps us further understand the relationship between the structure and function of BM2 channel at the atomic level.In addition,the results indicated that the polar environment formed by serine triad is the key factor of affecting the function of BM2 channel,which might gain meaningful information for the design of antiinfluenza drugs.2.The effects of the membrane environment on the conformation and hydration of BM2 channelIn order to clarify the mechanism of negative lipids and cholesterol regulating the function of BM2 channel,we constructed the models of different membrane environments complex with BM2 channel,and studied the conformational changes and hydration of BM2 channel in different membrane environments by molecular dynamics simulations.The results showed that negative lipids caused the increase of membrane order and thickness.Therefore,there is more space for opening BM2 channel.Additionally,the stable hydrogen bond networks between negative lipids and Cterminal portion of BM2 channel could maintain the open states of BM2 channel.The calculation results of water density of the channel showed that the presence of negative lipids enhanced channel hydration.We also constructed the models of cholesterol with different concentrations complex with BM2 channel to study the effects of cholesterol on the conformations and hydration of BM2 channel.The results showed that as the concentration of cholesterol in the membrane increased,membrane was more ordered and thicker,and the lipid packing was tighter.Thus,the channel would have more space to open and the channel hydration increased.This work indicated membrane environment is the vital factor of conformation and channel hydration of BM2,and clarified the microscopic process of membrane environment regulating the function of BM2,which enriched our understanding of the relationship between structure and function of BM2 channel.3.The interaction of proton channel blocker Amantadine and BM2 channelBM2 channel with highly conserved functional domain is regarded as an effective target against influenza virus B.However,effective inhibitors targeting BM2 channel have been absent until now.In order to deeply understand the detailed structural features of BM2 channel relevant to drug design,in this work,we performed multiple molecular dynamics simulations(including Classical Molecular Dynamics(CMD)simulations,Adaptive Streed Molecular Dynamics(ASMD)simulations and accelerated Molecular Dynamics(a MD)simulations)to explore the interactions of the proton channel blocker Amantadine and BM2 channel in detail.Our results indicated that Amantadine bound to BM2 channel follows a “bind and flip mechanism”.Amantadine mainly adopts up binding mode in the N-terminal portion of BM2 channel.When Amantadine adopts down binding mode,driven by the electrostatic repulsion induced by the positively charged C-terminal portion of BM2 channel,the proton channel blocker Amantadine will flip from down binding mode to up binding mode.In addition,we discovered a new key factor to explain ineffective inhibition of the proton channel blocker Amantadine to BM2 channel because of the unmatched spatial geometry between Amantadine and BM2 channel.Meanwhile,in this work,we also clarify the factors should be considered for the design of potent inhibitor targeting BM2 channel.Our work could enrich the structural feature information of BM2 channel and provide a theoretical basis for rational drug design targeting BM2 channel.