新型MFS-MDR转运蛋白MdrP晶体结构解析及其多药物外排的分子转运机制研究

前期研究证实，MdrP与已鉴定MFS超家族成员同源性较低，为MFS超家族一个新成员。功能鉴定表明，该蛋白既可独立偶联质子实现多药物外排，也可偶联Na+显著提高其活性。然而，MdrP如何通过Na+/质子驱动MdrP的构象改变，进而实现多药物外排的分子机制仍然未知。申请人旨在通过MdrP原核表达和X衍射技术对其晶体结构予以解析，以揭示Na+及质子驱动该蛋白构象转变的结构基础；在此基础上，通过定点突变，结合功能互补和活性鉴定、质子释放及ITC等实验，鉴定MdrP负责结构稳定及药物、质子或Na+结合的重要残基，明确其质子和Na+转位途径及多药物结合位点，最终揭示MdrP的多药物外排分子机制。作为首个偶联Na+的MFS-MDR转运蛋白，本项目的开展不仅可揭示MFS超家族转运蛋白新型的多药物外排分子机制，而且可为人们基于MDR转运蛋白离子结合位点为药物靶点的抗微生物药物研制提供新的思路与启示。

Preliminary experiments showed that MdrP shares quite low identity with characterized major facilitator superfamily (MFS) transporters, indicating that this transporter is a novel MFS member. Functional analysis reveals that MdrP can achieve H+-coupled mutltidrug efflux activity independently, and its mutltidrug efflux activity also can be significantly enhanced. However, it is unclear how the conformational switch of MdrP is driven by the coupling to proton and Na+ to exhibit multidrug efflux activity. In this project, we plan to analyze the crystal structure of MdrP through the combination of prokaryotic over-expression with X-ray diffusion technique and discover the structural basis of conformational change driven by the coupling to proton and Na+. On the basis of the above results, we plan to carry out site-directed mutagenesis of amino acid residues, and functional complementation tests combined with activity assays for Na+/H+ antiport and multidrug efflux. Based on the above experiments, we will identify the important residues for the stability of extracellul ar-facing conformation, drug binding, H+ binding and Na+ binding, respectively. And we further plan to carry out H+ release and isothermal titration calorimetry (ITC) experiments using purified MdrP and variants to find out the ionic pathway for proton and Na+ and clarify the drug-binding sites, respectively. As the first Na+-coupled MFS-MDR transporter, the implementation of this project will be helpful to discover a novel mechanism of MFS transporters for H+ coupling and Na+ coupling to multidrug efflux, and also positively contribute to the recognizition of cationic binding sites as drug targets of MDR transporters for the development of antimicrobial drugs.