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The University of Science and Technology has made progress in the study of the structure and function of multi-drug resistance proteins in human cancer cells

2023/7/24 Viewed：

Drug therapy (chemotherapy, targeted or immune drugs) is a very important means of cancer treatment. But over time, cancer cells develop resistance to almost all drugs, ultimately leading to treatment failure. Multidrug resistance ABC transporter-mediated drug efflux is a common mechanism of drug resistance in cancer cells. ABC transporters have a broad substrate spectrum and can expel many exogenous chemicals from the cell, including vinca alkaloids, podophylloids, anthracyclines, taxanes, and kinase inhibitors. Therefore, in the process of first-line clinical chemotherapy, due to their efflux of drugs, the therapeutic effect is greatly weakened or even disappeared.

Human ABC transporter C subfamily member ABCC3 (also known as multi-drug resistant protein MRP3) is widely expressed in various tissues, such as the adrenal gland, liver, pancreas, gallbladder, and small intestine. It has a wide range of substrate specificity, and in addition to transporting exogenous drugs that lead to drug resistance, it is also capable of transporting various endogenous metabolites such as glucuronic acid bilirubin, bile acids, and steroid hormones. Therefore, ABCC3 is involved in a variety of physiological processes in the body and is closely associated with human diseases, such as intrahepatic cholestasis of pregnancy (ICP) caused by the accumulation of bile acids and estrogens in cells.

Recently, the research team of Professor Chen Yuxing and Professor Zhou Congzhao from the School of Life Sciences and Medicine, University of Science and Technology of China, The protein structures of ABCC3 without ligand binding (apo-form) and physiological substrates Estradiol glucuronate (E217βG) and dehydroepiandrosterone sulfate (DHEAS) were analyzed by single particle cryo-electron microscopy. The overall resolutions were 3.1, 3.7 and 3.5 A, respectively. Based on the results of protein structure analysis and biochemical analysis, the authors found that ABCC3 has a biparental substrate-binding pocket, which is occupied asymmetrically by two conjugated hormone molecules. Combined with previous literature reports, the authors proposed the common characteristics of the binding pockets of similar multidrug-resistant protein substrates, which provided the direction for the rational design of inhibitors of MRPs. the relevant research results are presented as "Placing steroid hormones within the human ABCC3 transporter reveals a compatible amphiphilic substrate-binding. pocket "was published online in TheEMBO Journal on July 24, 2023.

Structural analysis showed that the apo-form ABCC3 structure presented an inward-facing conformation: the two nucleotide-binding domains (NBD) separated from each other, and the two transmembrane domains (TMD) formed a "V-shaped" substrate transport channel open toward the cytoplasmic matrix. Unlike the classic ABC transporter, the n-terminal has an additional five-strand TMD0 domain composed of transmembrane helices. The five strands of TMD0 spiral together across the membrane to form a compact and independent domain (Figure 1). The second transmembrane helix (TM) TM2 on TMD0 interacts with the core domain by forming an interaction interface of about 1700A2 with TM6 on TMD1. However, the detection of ATPase activity and radiosubstrate transport of ABCC3 showed that the TMD0 domain was not necessary for the transport function of ABCC3, and it was speculated that it might affect the localization of ABCC3 on polarized cells or interact with other proteins.

Figure 1: apo-form ABCC3 structure

The inward-facing conformation of ABCC3 binding substrate E217βG was still adopted, and the local rearrangement of key residues in the substrate pocket helped the binding and fixation of E217βG molecules. In this structure, two E217βG molecules are cooperatively bound inside ABCC3 in a "V-shape" in space. The hydrophobic steroid nuclei of the E217βG molecule occupy the hydrophobic pocket and coalesce into the "V-shaped" vertex, and the two hydrophilic glucuronic acid groups are located in the polar pockets formed by the alkaline residues on both sides of TMD1 and TMD2, respectively (Figure 2). Biochemical experiments have shown that two basic residues, Arg1193 and Arg1245, located on the side of TMD1, are critical for the binding of E217βG molecules, and their mutation leads to the complete loss of substrate-stimulated ATPase activity, indicating that the long side chain of arginine and strong alkalinity are necessary for substrate binding.

Figure 2: E217βG substrate binding pocket

Although DHEAS and E217βG have a similar hydrophobic steroid, the glucuronic acid group of E217βG is located at the 17 'position of the steroid, while the sulfonic acid group of DHEAS is located at the 3' position of the steroid. In the DHEAS-bound ABCC3 structure, there is a similar "V-shaped" binding pattern (Figure 3), but compared to E217βG, the steroidal nuclei of the DHEAS molecule are in the opposite direction, thus enabling the sulfonic acid groups to also occupy the alkaline pockets on both sides of TMD1 and TMD2. Its sulfonic acid group forms salt bonds with Arg1193 and Arg124, two arginines conserved on TMD1, and biochemical activity experiments show that these polar interactions are important for the recognition of DHEAS.

Figure 3: DHEAS binding sites

The authors further analyzed the reported MRPs structures and their substrates and found that they all have a negatively charged group conjugated to a hydrophobic core structure. The substrate binding pocket of ABCC3 has a large hydrophobic chamber in addition to two positively charged hydrophilic regions, so it is just right for substrate binding. There is a cluster of alkaline residues on one side of TMD1 and it is highly conserved in MRPs. In contrast, the hydrophilic region on the side of TMD2 is less positive and less conservative. Therefore, the authors divided the substrates of MRPs into three categories according to the size of substrates and the different conjugation methods, and proposed corresponding binding methods of substrates (Figure 4). In conclusion, this study not only elucidates the molecular mechanism of ABCC3-specific substrate recognition, but also proposes a substrate binding pattern common to all MRPs, further broadening our understanding of the molecular mechanisms of multidrug resistance.

Figure 4: MRPs substrate binding pattern

Professor Yuxing Chen, Professor Congzhao Zhou and Associate Professor Wentao Hou from the University of Science and Technology of China are co-corresponding authors, and doctoral student Jie Wang is the first author of the paper. Cryo-electron microscopy data collection was completed at the Beijing Institute of Biophysics, Chinese Academy of Sciences and the Integrated Imaging Center, University of Science and Technology of China. The research was funded by the Ministry of Science and Technology, the National Natural Science Foundation of China, the Chinese Academy of Sciences and the University of Science and Technology of China.

Original link：https://doi.org/10.15252/embj.2022113415

(Department of Life Sciences and Medicine, Department of Scientific Research)

Source: China University of Science and Technology News

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