人源多药耐药相关蛋白MRP4与MRP5的结构和转运机制研究

多药耐药相关蛋白（multidrug resistance-associated protein，MRP），属于ATP结合盒（ABC）转运蛋白的ABCC亚家族成员。人源多药耐药相关蛋白4（hMRP4，也称为ABCC4）和多药耐药相关蛋白5（hMRP5，也称为ABCC5）具有MRP蛋白典型拓扑结构，并在膜上转运各种底物，包括抗癌和抗病毒药物分子，驱动多药耐药性的发生。然而，由于缺乏高分辨率结构，hMRP4和hMRP5的潜在转运机制仍不清楚。在本论文研究中，我们使用冷冻电子显微镜（cryo-EM）解析了hMRP4和hMRP5多个不同状态的相对较高分辨率三维结构，为研究hMRP4和hMRP5的多药耐药机制提供了精准的结构基础。

在hMRP4的研究中，我们获得了apo、前列腺素E1（PGE1）结合、及舒林酸（sulindac）结合的三个向内开放的hMRP4结构；此外，我们还获得了结合ATP的hMRP4向外开放的近原子冷冻电镜结构。我们通过分子动力学模拟、生化等实验证明PGE1、sulindac能够与hMRP4稳定结合；同时，基于上述的结构分析比较，我们进一步发现抑制剂sulindac与底物PGE1竞争性结合hMRP4转运通道中的同一个疏水结合口袋。通过上述四个hMRP4的结构比较和生化分析，我们明确了hMRP4的工作模型，并对sulindac抑制hMRP4转运PGE1底物的分子机制进行明确阐释。

在hMRP5的研究中，我们获得了野生型hMRP5（wt-hMRP5）相对较高分辨率冷冻电子显微镜结构，该结构呈典型的向内开放构象并发现来源于hMRP5氨基端（N-terminal）的多肽（C46-S64）直接结合在hMRP5的转运通道中；因此，我们首次获得了一个来自氨基酸多肽自抑制的ABC转运蛋白结构。我们接着分析了C46-S64作用于hMR5的结构基础，并通过分子动力学模拟、点突变、亲和力分析等实验证实C46-S64抑制hMRP5的分子机制。进一步，我们构建了hMRP5的N端截短体（hMRP5-Δ1-94）并解析hMRP5-Δ1-94的相对较高分辨率冷冻电镜结构。与我们推测一致，该冷冻电镜结构呈现向内开放的构象，且在其转运通道中没有观察到多余的密度，这进一步说明我们捕获到apo构象的hMRP5结构。基于N端多肽与hMRP5结合的结构信息，我们设计不同的肽段并通过微量热泳动（MST）分析肽段与hMRP5之间的结合亲和力，最终筛选出一段与hMRP5有高亲和力的肽段（M5PI）。有意思的是，通过结合亲和力与爪蟾卵母细胞体系转运实验，我们不仅证明了M5PI与hMRP5结合的特异性，还验证了M5PI抑制hMRP5外排抗肿瘤药物阿霉素的功能。同样，我们通过冷冻电镜解析了hMRP5与M5PI结合的复合物结构。通过比对wt-hMRP5和M5PI结合hMRP5的结构，发现M5PI与hMRP5结合位点、C46-S64与hMRP5结合的位点大致相同，两者均通过疏水作用力和盐桥稳定在hMRP5的底物结合口袋。此外，我们还解析了抑制剂曲喹辛（trequinsin）与hMRP5结合的复合物结构；通过结构分析及分子动力学模拟等实验证实trequinsin能稳定结合在hMRP5的结合口袋处。通过不同状态下的hMRP5结构比对，发现trequinsin占据的口袋与C46-S64/M5PI存在空间位置的差异，这表明M5PI与trequinsin具有不同的抑制机制，M5PI可能在对hMRP5的抑制作用上与trequinsin有一定的互补作用。

综上所述，我们通过冷冻电镜、分子动力学模拟、及生物化学测定等技术手段，揭示了hMRP4和hMRP5底物转运和抑制机制的结构基础，尤其是首次发现氨基端多肽自抑制的hMRP5构象，并提出了一个新的hMRP5转运构象循环和工作模型。总的来说，该研究增强了我们对多药耐药相关蛋白转运机制的理解；同时，后续开发多药耐药蛋白特异性抑制剂提供了直接线索，对开发靶向药物具有启示意义；另外，这项研究极大的拓展了我们对ABC转运蛋白的结构动力学和抑制作用的理解。

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