Copper transporting P-type (P_1B-1-) ATPases are essential for cellular homeostasis. Nonetheless, the E1-E1P-E2P-E2 states mechanism of P_1B-1-ATPases remains poorly understood. In particular, the role of the intrinsic metal binding domains (MBDs) is enigmatic. Here, four cryo-EM structures and molecular dynamics simulations of a P_1B-1-ATPase are combined to reveal that in many eukaryotes the MBD immediately prior to the ATPase core, MBD⁻¹, serves a structural role, remodeling the ion-uptake region. In contrast, the MBD prior to MBD⁻¹, MBD⁻², likely assists in copper delivery to the ATPase core. Invariant Tyr, Asn and Ser residues in the transmembrane domain assist in positioning sulfur-providing copper-binding amino acids, allowing for copper uptake, binding and release. As such, our findings unify previously conflicting data on the transport and regulation of P_1B-1-ATPases. The results are critical for a fundamental understanding of cellular copper homeostasis and for comprehension of the molecular bases of P_1B-1-disorders and ongoing clinical trials.

铜转运 P 型 (P _1B-1 -) ATP 酶对于细胞稳态至关重要。尽管如此，E1-E1P-E2P-E2 状态 P _1B-1 -ATP 酶的机制仍然知之甚少。特别是，内在金属结合域（MBD）的作用是神秘的。在这里，P _1B-1 -ATPase 的四种冷冻电镜结构和分子动力学模拟相结合，揭示了在许多真核生物中，紧邻 ATPase 核心之前的 MBD MBD ^-1发​​挥着结构作用，重塑了离子摄取地区。相反，MBD ^-1 、MBD ^-2之前的MBD可能有助于将铜递送至ATP酶核心。跨膜结构域中不变的 Tyr、Asn 和 Ser 残基有助于定位提供硫的铜结合氨基酸，从而允许铜的摄取、结合和释放。因此，我们的研究结果统一了先前关于 P _1B-1 -ATP 酶的运输和调节的相互矛盾的数据。这些结果对于从根本上理解细胞铜稳态、理解 P _1B-1疾病的分子基础以及正在进行的临床试验至关重要。