Structure meets function for OATP1B1, a transporter involved in the uptake of endogenous and xenobiotic materials and drugs

项目总结 多态转运蛋白OATP1B1在肝脏摄取和处置 内源性分子和药物。此外，OATP1B1还参与了许多药物-药物相互作用(DDiS)。 它的多面性。尽管经过几十年的研究，许多因素与配体结合的决定因素有关， 运输机制和能量学，以及这一重要运输器的结构尚未解决。这个 长期目标是在微观和宏观水平上描述肝脏运输的特征。由于 OATP1B1在肝脏清除中的功能重要性，这是第一个研究的目标。中心假设是 人类OATP1B1的结构和对分离蛋白进行的功能分析将明确定义 配体结合、运输和抑制的机制。这项研究的基本原理是 蛋白质结构与明确的、可重复性的活性分析相结合是完全必要的 描述运输机制，并解决文献中长期存在的相互矛盾的数据。 建立人OATP1B1的强健表达和纯化方案有助于实现这两个目标 具体目标：1)用低温电子显微镜解析OATP1B1的结构，并用分子动力学模拟分析它们的运动。2) 从功能上表征OATP1B1的转运机制。对于第一个目标，最优条件是 结果OATP1B1的Cryo-EM图像质量最高，数据采集在 配基的存在和不存在。低温电磁研究将通过MD模拟和对接得到加强 将揭示仅靠结构分析无法获得的细节的研究。不同的生物化学 条件(apo和holo)增加了A)求解各种结合的结构决定因素的可能性 OATP1B1的两个配基结合部位的配基(具有不同的化学成分)。B)解决多项式的结构 作为运输周期一部分的构象。对于第二个目的，我们将使用重组的纯化蛋白 蛋白脂质体用于表征OATP1B1的固有属性，例如为运输提供能量的离子， 膜电压、pH值的影响。这些结果将与细胞分析进行比较， 包括完整的细胞机械，以及可能直接或间接影响的其他质膜转运体 OATP1B1。申请者认为，本申请中提出的研究是创新的，因为它 介绍了两种从未被探索用于任何OATP异构体的方法。这些可能会揭示一种 对OATP1B1结构/功能关系的大量新见解。拟议的研究是 意义重大，因为OATP1B1涉及几种常用处方药的DDIS，有时会导致 有生命危险的情况。这项研究将揭开与OATP1B1和OATP1B1结合的确切决定因素 运输。最终，这里产生的结构和功能知识有可能帮助设计 显示出DDIS倾向较低的药物。

Project summary The polymorphic transporter OATP1B1 plays significant roles in the hepatic uptake and disposition of endogenous molecules and drugs. Further, OATP1B1 is involved in numerous drug-drug interactions (DDIs) due to its multispecificity. Despite decades of research, many factors related to the determinants of ligand binding, transport mechanism and energetics, as well as the structure of this important transporter are unresolved. The long-term goal is to characterize hepatic transport at the microscopic and macroscopic levels. Due to the functional importance of OATP1B1 in liver clearance, this is the first target studied. The central hypothesis is that structures of human OATP1B1 and functional assays performed on isolated proteins will unequivocally define the mechanism of ligand binding, transport, and inhibition. The rationale that underlies this research is that protein structures coupled with unambiguous, reproducible activity assays are absolutely required to fully characterize the mechanism of transport and resolve long-standing conflicting data reported in the literature. Establishing robust expression and purification protocols for human OATP1B1 facilitates pursuing the two Specific Aims: 1) Resolve structures of OATP1B1 by Cryo-EM and analyze their motions by MD simulations. 2) Functionally characterize the transport mechanism of OATP1B1. For the 1st Aim, the optimal conditions that result in the highest image quality for Cryo-EM of OATP1B1 would be screened, and data would be acquired in the presence and absence of ligands. The Cryo-EM studies will be augmented by MD simulations and docking studies that would reveal details that are not accessible by mere structural analysis. The different biochemical conditions (apo and holo) increase the likelihood of A) solving the structural determinants of the binding of various ligands (with distinct chemistries) to the two ligand-binding sites of OATP1B1. B) solving the structures of multiple conformations that are part of the transport cycle. For the 2nd Aim, we will use purified protein reconstituted into proteoliposomes to characterize the inherent properties of OATP1B1 such as the energizing ion for transport, the influence of the membrane voltage, and pH. Those results would be compared with cellular assays that include intact cellular machinery, and other plasma membrane transporters that might directly or indirectly affect OATP1B1. The research proposed in this application is innovative, in the applicant’s opinion because it introduces two methodologies that have never been explored for any OATP isoform. These could uncover a plethora of novel insights into the structure/function relationship of OATP1B1. The proposed research is significant since OATP1B1 is involved in DDIs of several commonly prescribed drugs, sometimes resulting in life-threatening situations. This study would unravel the exact determinants of ligand binding to OATP1B1 and transport. Ultimately, the structural and functional knowledge generated here has the potential to aid in the design of drugs that exhibit a lower propensity for DDIs.