P-糖蛋白 (P-gp) 在细胞解毒和药物流出过程中发挥着至关重要的作用，在向内 (IF) 开放、闭塞和向外 (OF) 状态之间转换以促进底物转运。它在癌症治疗中发挥着至关重要的作用，P-gp 会导致多药耐药表型。在我们的研究中，进行了经典和增强的分子动力学（MD）模拟来剖析 P-gp 构象态的结构和功能特征。我们先进的分子动力学模拟，包括动力学激发靶向分子动力学 (ketMD) 和绝热偏置分子动力学 (ABMD)，为状态转换和易位机制提供了更深入的见解。我们的研究结果表明，TM4 和 TM10 螺旋的解开是正确实现外向构象的先决条件。以扭结的 TM4 和 TM10 螺旋为特征的 IF 闭塞构象的模拟一致证明跨膜结构域 (TMD) 和核苷酸结合结构域 2 (NBD2) 之间的通讯发生改变，表明该界面在抑制 P-gp 的外排功能中的含义。特别强调连接 NBD1 和 TMD2 的非结构化连接片段及其在转运蛋白动态中的作用。由于存在连接子，我们特别注意到胆固醇 (CHOL) 可能通过 TM4-TM6 门户进入，从而揭示了参与调节 CHOL 的关键残基。因此，我们建议这种进入机制可用于某些 P-gp 底物或抑制剂。我们的结果为了解 P-gp 功能和开发新的 P-gp 抑制剂提供了关键数据，从而建立更有效的对抗多药耐药性的策略。© 2024 作者。

P-glycoprotein (P-gp) plays a crucial role in cellular detoxification and drug efflux processes, transitioning between inward-facing (IF) open, occluded, and outward-facing (OF) states to facilitate substrate transport. Its role is critical in cancer therapy, where P-gp contributes to the multidrug resistance phenotype. In our study, classical and enhanced molecular dynamics (MD) simulations were conducted to dissect the structural and functional features of the P-gp conformational states. Our advanced MD simulations, including kinetically excited targeted MD (ketMD) and adiabatic biasing MD (ABMD), provided deeper insights into state transition and translocation mechanisms. Our findings suggest that the unkinking of TM4 and TM10 helices is a prerequisite for correctly achieving the outward conformation. Simulations of the IF-occluded conformations, characterized by kinked TM4 and TM10 helices, consistently demonstrated altered communication between the transmembrane domains (TMDs) and nucleotide binding domain 2 (NBD2), suggesting the implication of this interface in inhibiting P-gp's efflux function. A particular emphasis was placed on the unstructured linker segment connecting the NBD1 to TMD2 and its role in the transporter's dynamics. With the linker present, we specifically noticed a potential entrance of cholesterol (CHOL) through the TM4-TM6 portal, shedding light on crucial residues involved in accommodating CHOL. We therefore suggest that this entry mechanism could be employed for some P-gp substrates or inhibitors. Our results provide critical data for understanding P-gp functioning and developing new P-gp inhibitors for establishing more effective strategies against multidrug resistance.© 2024 The Authors.