Structural basis of ion uptake in copper-transporting P1B-type ATPases

Copper is an essential micronutrient that cycles between Cu⁺ and Cu²⁺ to support key enzymes but is toxic when unregulated, necessitating tight intracellular control via chaperones and export systems. P-type ATPases couple ATP hydrolysis to transport across membranes and include the P1B subclass specialized for transition metal efflux, with human ATP7A and ATP7B as clinically important members. Despite established alternating access E1 (inward-facing) and E2 (outward-facing) states in P-type ATPases, no E1-state structure had been available for P1B-ATPases, obscuring basic mechanisms of ion uptake, transfer from cytosolic chaperones, and formation of high-affinity copper-binding sites. This study aims to determine an inward-facing E1 conformation of a copper-transporting P1B-ATPase and elucidate the structural basis of Cu⁺ uptake and delivery from copper chaperones.

Prior structural knowledge for P1B-ATPases was limited to E2P and E2·P conformations from L. pneumophila CopA (LpCopA), X. tropicalis ATP7B, and S. sonnei ZntA, as well as isolated soluble domains and a low-resolution cryo-EM model of A. fulgidus CopA (AfCopA). Proposed mechanisms included recruitment of Cu chaperone CopZ via an electropositive platform (MB′) and models of Cu⁺ entry involving residues M158, E205, D336, and the CPC motif on M4. However, direct structural evidence for an E1, metal-uptake state and the precise molecular determinants of chaperone-mediated Cu⁺ transfer to the ATPase core were lacking. Comparisons to P2-type ATPases (e.g., SERCA) show conservation of overall reaction cycle but suggested potential differences in domain architecture and transitions in P1B members.

The authors expressed a truncated A. fulgidus CopA lacking N- and C-terminal heavy metal binding domains (AfCopAΔNAC) in E. coli and purified it in detergent. Crystallization used the HiLiDe approach with DOPC and C12E8, in the presence or absence of CuSO4, yielding crystals diffracting to 2.7–2.8 Å. X-ray diffraction data were collected at the Swiss Light Source. The structure was solved by molecular replacement (Phaser) using AfCopA soluble domains and LpCopA M-domain as search models, followed by model building (Coot), Rosetta and Phenix refinement. Complementary data were collected at the Cu absorption edge (1.37 Å; 3.3 Å resolution) with MR-SAD phasing to probe Cu⁺ binding. Ensemble refinement (Phenix) assessed conformational heterogeneity. Molecular dynamics simulations (CHARMM force field, membrane-embedded models) explored Cu⁺ coordination at the proposed entry site and transfer toward the transmembrane binding region. Rigid-body docking of a homology model of AfCopZ (based on E. hirae CopZ) to the E1 AfCopA structure used pyDockWEB to examine chaperone-ATPase interactions. Functional assays included reconstitution into liposomes and Cu²⁺-stimulated ATPase measurements at 65 °C (colorimetric inorganic phosphate assay), and ICP-MS to determine Cu binding stoichiometry for wild-type and alanine mutants (entry-site and M4–M6 conserved residues). Mutagenesis used QuikChange. Data analyses included cavity mapping (HOLLOW) and structural alignments to SERCA and KdpB.

• Determined an inward-facing E1 structure of AfCopA at up to 2.7 Å resolution (R/Rfree 22.5/25.7), revealing a unique cytosolic domain arrangement distinct from SERCA but reminiscent of P1A KdpB, including a ~100° counterclockwise rotation of the A-domain relative to the P-domain during the E2→E1 transition.
• The M-domain partitions into two helix bundles (MA–M2 and M3–M6) that move relative to each other, closing the outward pathway and opening the cytosolic side. The conserved CPC motif (M4) becomes exposed to the cytosol in E1.
• Residues critical for high-affinity binding (CPC on M4; Y682–N683 on M5; M711–S714–S715 on M6) are oriented toward the M4–M6 core in E1, with M711 repositioned from a membrane-exposed E2·P location.
• Functional assays: WT AfCopAΔNAC showed Cu⁺-stimulated turnover of 242 ± 26 nmol Pi mg⁻¹ min⁻¹. Alanine mutations of M4–M6 conserved residues (C380, C382, Y682, N683, M711, S714, S715) severely impaired or abolished activity, whereas mutations in the proposed entry region (M158, E205, D336) had modest effects under free Cu conditions.
• ICP-MS revealed Cu binding stoichiometry of 0.87 ± 0.10 Cu per AfCopAΔNAC. Binding was markedly reduced by mutations in CPC (C380A, C382A) and M5/M6 motifs (Y682A, N683A, M711A, S714A, S715A), consistent with required transmembrane ligands.
• Cu-edge anomalous data detected a site near C382 in the CPC motif with refined ~60% occupancy, supporting a transient Cu⁺ entry site comprising M158 and CPC cysteines.
• Ensemble refinement indicated conformational flexibility around the CPC region and M158, consistent with a dynamic entry site.
• MD simulations supported transient trigonal-planar Cu⁺ coordination by M158, C380, C382 and rapid transfer toward M711 with coordinating waters, suggesting a pathway from entry to a single high-affinity site in later E1 states.
• Docking positioned CopZ in a groove near the electropositive MB′ platform, placing CopZ-bound Cu⁺ within ~6 Å of certain M158 conformations, supporting direct Cu⁺ transfer from CopZ to M158, followed by handover to the CPC and internal site(s).

The E1 AfCopA structure addresses the long-standing gap in understanding how P1B-ATPases acquire Cu⁺ from cytosolic donors. The unique A-domain arrangement and separation into two M-domain sub-bundles create a cytosolic-accessible CPC region compatible with chaperone interaction. The data unify previous models by proposing that CopZ docks electrostatically at MB′ and directly transfers Cu⁺ to M158, forming a transient mixed donor site with CopZ CxxC and M158. The methionine then reorients to cooperate with CPC cysteines, forming a transient entry site from which the ion proceeds to a high-affinity transmembrane site formed by CPC and M5/M6 residues (including M711). Mutational, ICP-MS, anomalous scattering, ensemble refinement, and MD collectively corroborate this mechanism. The observed A-domain rotation and linker constraints suggest mechanistic differences from P2-type ATPases, offering a P1-specific view of the E2·P→E1 transition. The findings likely extend to human ATP7A/B, informing how copper is delivered and occluded prior to ATP-driven transitions.

This work presents the first high-resolution inward-facing E1 conformation of a copper-specific P1B-type ATPase (AfCopA), revealing an A-domain arrangement and M-domain reorganization that enable chaperone docking and Cu⁺ entry. Evidence supports a sulfur-rich transient entry site involving M158 and CPC cysteines, direct Cu⁺ transfer from CopZ, and subsequent progression to a single high-affinity site in later E1 states. These insights clarify the structural basis of ion uptake in P1B-ATPases and are relevant to human ATP7A/B. Future work should determine metal-bound later E1 intermediates and, ideally, structures of ATPase–chaperone complexes to resolve the architecture of the high-affinity site(s) and the precise sequence of transfer events.

• Metal occupancy and identity in the high-affinity transmembrane site(s) could not be unambiguously assigned in the high-resolution data; anomalous signal near the CPC indicates a transient entry site with partial occupancy (~60%).
• The structure represents an early E1 state and uses a truncated construct lacking N- and C-terminal heavy metal binding domains, which may influence regulation and docking interactions.
• No experimental co-structure with CopZ was obtained; chaperone docking is inferred computationally.
• Electron density is locally weak and conformationally heterogeneous around the CPC/M158 region, reflecting dynamics that complicate precise side-chain assignments.
• Functional assays used conditions with free Cu donors; roles of entry-site residues may be more prominent under physiological chaperone-delivered Cu⁺.
• Generalization to human ATP7A/B, while plausible, requires direct validation.