Mutational analysis reveals the importance of residues of the access tunnel inhibitor site to human P-glycoprotein (ABCB1)-mediated transport.

IF 4.5 3区 生物学 Q1 BIOCHEMISTRY & MOLECULAR BIOLOGY
Protein Science Pub Date : 2024-09-01 DOI:10.1002/pro.5155
Paula B Salazar, Megumi Murakami, Nandhini Ranganathan, Stewart R Durell, Suresh V Ambudkar
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引用次数: 0

Abstract

Human P-glycoprotein (P-gp) utilizes energy from ATP hydrolysis for the efflux of chemically dissimilar amphipathic small molecules and plays an important role in the development of resistance to chemotherapeutic agents in most cancers. Efforts to overcome drug resistance have focused on inhibiting P-gp-mediated drug efflux. Understanding the features distinguishing P-gp inhibitors from substrates is critical. Cryo-electron microscopy has revealed distinct binding patterns, emphasizing the role of the L-site or access tunnel in inhibition. We substituted 5-9 residues of the L-site with alanine to investigate whether the binding of a second inhibitor molecule to the L-site is required for inhibiting drug efflux. We reveal, for the first time, that mutations in the L-site affect the drug efflux activity of P-gp, despite their distance from the substrate-binding pocket (SBP). Surprisingly, after the mutations were introduced, inhibitors such as tariquidar and zosuquidar still inhibited drug efflux by mutant P-gps. Communication between the transmembrane helices (TMHs) and nucleotide-binding domains (NBDs) was evaluated using the ATPase assay, revealing distinct modulation patterns by inhibitors for the mutants, with zosuquidar exhibiting substrate-like stimulation of ATPase. Furthermore, L-site mutations abolished ATP-dependent thermal stabilization. In silico molecular docking studies corroborated the altered inhibitor binding due to mutations in the L-site residues, shedding light on their critical role in substrate transport and inhibitor interactions with P-gp. These findings suggest that inhibitors bind either to the SBP alone, and/or to alternate site(s) when the L-site is disabled by mutagenesis.

突变分析揭示了通路隧道抑制位点残基对人P-糖蛋白(ABCB1)介导的转运的重要性。
人类 P 糖蛋白(P-gp)利用 ATP 水解产生的能量将化学性质不同的两性小分子药物外流,在大多数癌症的化疗药物耐药性产生过程中发挥着重要作用。克服耐药性的方法主要是抑制 P-gp 介导的药物外流。了解区分 P-gp 抑制剂和底物的特征至关重要。冷冻电镜显示了不同的结合模式,强调了 L 位点或通道在抑制中的作用。我们用丙氨酸取代了 L 位点的 5-9 个残基,以研究抑制药物外流是否需要第二个抑制剂分子与 L 位点结合。我们首次发现,L 位点的突变会影响 P-gp 的药物外流活性,尽管它们与底物结合口袋(SBP)相距甚远。令人惊讶的是,在引入突变后,tariquidar 和 zosuquidar 等抑制剂仍能抑制突变 P-gps 的药物外流。利用 ATPase 试验评估了跨膜螺旋(TMHs)和核苷酸结合域(NBDs)之间的交流,结果显示抑制剂对突变体的调节模式各不相同,其中 zosuquidar 对 ATPase 的刺激类似于底物。此外,L-位点突变取消了 ATP 依赖性热稳定。硅学分子对接研究证实了 L 位点残基突变导致的抑制剂结合改变,揭示了它们在底物转运和抑制剂与 P-gp 相互作用中的关键作用。这些发现表明,当 L 位点因突变而失效时,抑制剂要么单独与 SBP 结合,要么与替代位点结合。
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来源期刊
Protein Science
Protein Science 生物-生化与分子生物学
CiteScore
12.40
自引率
1.20%
发文量
246
审稿时长
1 months
期刊介绍: Protein Science, the flagship journal of The Protein Society, is a publication that focuses on advancing fundamental knowledge in the field of protein molecules. The journal welcomes original reports and review articles that contribute to our understanding of protein function, structure, folding, design, and evolution. Additionally, Protein Science encourages papers that explore the applications of protein science in various areas such as therapeutics, protein-based biomaterials, bionanotechnology, synthetic biology, and bioelectronics. The journal accepts manuscript submissions in any suitable format for review, with the requirement of converting the manuscript to journal-style format only upon acceptance for publication. Protein Science is indexed and abstracted in numerous databases, including the Agricultural & Environmental Science Database (ProQuest), Biological Science Database (ProQuest), CAS: Chemical Abstracts Service (ACS), Embase (Elsevier), Health & Medical Collection (ProQuest), Health Research Premium Collection (ProQuest), Materials Science & Engineering Database (ProQuest), MEDLINE/PubMed (NLM), Natural Science Collection (ProQuest), and SciTech Premium Collection (ProQuest).
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