Molecular Modeling Study On Work Mechanism Of PfMATE Transporter From Pyrococcus Furiosus

Multidrug and toxic compound extrusion(MATE) family transporters expel endogenous cationic, lipophilic substances and xenobiotic across cell membranes by dissipating a preexisting Na+ or H+ gradient. In cancer cells and bacterial pathogens, MATE transporters export structurally diverse anti-cancer drugs. MATE family transporters induce multiple-drug resistance(MDR) of bacterial pathogens and cancer cells, thus causing critical reductions in the therapeutic efficacies of antibiotics and anti-cancer drugs. Therefore, MATE has attracted broad attention as an important therapeutic target for MDR and the demand for the discovery of molecules capable of antagonizing the functions of MATE transporters is high because of their clinical importance. MATE inhibitors have long been awaited in the field of clinical medicine. However, due to little understanding about the atomic-level information of work mechanism of MATE transporters, the screening campaigns for MATE inhibitors leads have achieved very little success. In this thesis, molecular dynamics(MD) simulations were conducted to explore the conformational regulation mechanism,activity regulation mechanism of PfMATE transporter from Pyrococcus furiosus and inhibitory mechanism of a macrocyclic peptide MaD5 on PfMATE,the bind mode between macrocyclic peptides inhibitors and PfMATE transporter.The first part of the thesis is to explore the conformational regulation mechanism of PfMATE transporter from Pyrococcus furiosus based on different protonation state of Asp41. Two MD simulation systems were investigated: a system with protonation of Asp41 and a system without protonation of Asp41, which were named by D184(H)D41(H) system and D184(H) system, respectively. Firstly, MD simulation results indicate that conformational changes mainly happen in extracellular regions of PfMATE protein. Further analysis reveals that Pf MATE protein experiences different motion mode and forms different conformation based on different protonation state of Asp41. In the D184(H)D41(H) system, PfMATE experiences an opening motion and forms a more outward-open conformation. As for the D184(H) system, the protein has a anticlockwise rotational motion with the channel axis of protein and the more outward-open conformation does not appear. It can be inferred that protonation of Asp41 is essential for conformational regulation of PfMATE during transporting substrates.The second part of the thesis is to explore the activity regulation mechanism for PfMATE transporter based on the wild type and three single-mutation M173A/N180A/M206 A states. Firstly, PCA analysis and dynamic motion analysis indicate that PfMATE experiences different conformational ensembles in the M173A/N180A/M206 A states from the WT state and these single-mutations disturb the dynamic motion mode of PfMATE. Secondly, PMF analysis shows that protein in WT state has conformational transformation between different basins, which disappears in M173A/N180A/M206 A states. Finally, dynamical network analysis and DCCM analysis show that critical conformational communication pathways are disturbed in the M173A/N180A/M206 A states. It can be concluded that single-mutations M173A/N180A/M206 A disrupt the conformational regulation of PfMATE, providing an additional explanation for reason of the reduction of the transport activity in the three mutation states.The third part of the thesis is to investigate the inhibitory mechanism of a macrocyclic peptide Ma D5 on PfMATE. Firstly, PfMATE adopts a more out-open conformation and TM1 tends to bend in the apo system by comparing the conformational changes of the apo system and inhibitor-bound system during the simulation. However, in the inhibitor-bound system, the PfMATE experiences smaller conformational change, indicating the inhibitory activity of MaD5. Then, residue interaction network analysis shows that Ma D5 blocks the conformational regulation pathways of PfMATE transporter. Finally, binding interaction calculation indicates that interactions between the minicycle head of MaD5 and the binding pocket in the N-lope of PfMATE are mainly responsible for binding of MaD5. This study provides an important insight for the design of more potent macrocyclic peptide inhibitors.The forth part of the thesis is to further investigate interaction mechanism of macrocyclic peptide on PfMATE. Based on the complex structure of Mad5,MaD3 S and PfMATE,MD simulation study was conducted. MD simulation and binding free energy calculation help us understand the bind mode between macrocyclic peptide and PfMATE. Binding energy analysis shows that the interactions between polar residus of PfMATE and macrocyclic peptide play critical role in binding of macrocyclic peptide. Electrostatic interaction,hydrogen bond interaction are main interaction between polar residus of PfMATE and macrocyclic peptide. In this work, a more effective structural framework of macrocyclic peptide inhibitor is presented. Gln34 and Arg284 are the critical residues during design of more effective macrocyclic peptide inhibitor.Our molecular study results provide atom-level insight into detail information of work mechanism of MATE transporters and the inhibitory mechanism of macrocyclic peptide on PfMATE. These findings will be very meaningful to explore the MDR mechanism of PfMATE and provide important clues for the design of more potent macrocyclic peptide inhibitors.