Copper-transporting P-type ATPases use a unique ion-release pathway

Abstract

Heavy metals in cells are typically regulated by PIB-type ATPases such as the copper transporting Cu+-ATPases. The first crystal structure of a Cu+-ATPase (LpCopA) was trapped in a transition state of dephosphorylation (E2.Pi) and inferred to be occluded. The structure revealed a PIB-specific topology and suggested a copper transport pathway across the membrane. Here we show by molecular dynamics (MD) simulations that extracellular water solvates the transmembrane (TM) domain, indicative of a pathway for Cu+ release. Furthermore, a new LpCopA crystal structure determined at 2.8 Å resolution, trapped in the E2P state (which is associated with extracellular exchange in PII-type ATPases), delineates the same conduit as also further supported by site-directed mutagenesis. The E2P and E2.Pi states therefore appear equivalent and open to the extracellular side, in contrast to PII-type ATPases where the E2.Pi state is occluded. This indicates that Cu+-ATPases couple dephosphorylation differently to the conformational changes associated with ion extrusion. The ion pathway may explain why Menkes’ and Wilson’s disease mutations at the extracellular side impair protein function, and points to an accessible site for novel inhibitors targeting Cu+-ATPases of pathogens.

Membrane Anchoring and Ion-Entry Dynamics in P-type ATPase Copper Transport

“…Although efforts have been made to develop polarizable force fields (75,76), a complex membrane protein system is not the ideal test case, and such force fields might introduce artificial effects that would be hard or impossible to detect. Nevertheless, our previous simulations show that it is possible to obtain realistic results using nonpolarizable force-field parameters (10,11). In addition, a Cu þ -bound E1 state, high-resolution structure would add significantly to our understanding of the ion-entry process and internal coordination.…”

“…In addition, although the CHARMM36 force field is excellent for modeling atomistic lipid interactions that are essential to establish the lipid partitioning of LpCopA, it does not contain Cu þ parameters. Since parameterization of transition metal ions requires a major effort, we instead shifted to the ffGromos53a6 force field (45), which proved to be a successful approach for determining the structural dynamics and qualitative free energies associated with ion release from a Cu þ ATPase (10). Still, the inherent difficulty of performing a forcefield parameterization of metal ions should be acknowledged, and other copper-protein models have been described (73,74).…”

“…In addition to TM helices M1-M6 present in all P-type ATPases, the heavy-metal P IB -ATPases also contain two N-terminal class IB-specific structural features: 1) TM helices MA and MB, and 2) an N-terminal heavy-metalbinding domain (HMBD). Furthermore, a recent study using a combined crystallography/computational approach indicated that the P IB -specific TM helices contribute to a Cu þ -release pathway regulated by unique structural rearrangements (10). In contrast, the HMBD binds metal ions but does not seem to be strictly required for ion translocation, which suggests a regulatory function (9,21,22).…”

“…In the docked position, the chaperone Cu þ -binding site presumably is located close to the putative initial Cu þ receivers Met148, Glu205, and Asp337 (residue numbers refer to LpCopA throughout), as observed for Ca 2þ in the related Ca 2þ -transporting P-type ATPase SERCA1a (9,25). In SERCA1a, the ions are then transferred to two internal Ca 2þ -binding sites (26,27); however, the exact number, location, and chemical nature of the entry pathways in CopA proteins are unknown because the available structural information is limited to E2 states (9,10), where copper already has been deposited on the noncytosolic side. Mutagenesis and copper binding experiments have suggested two intramembranous ion-binding sites for AfCopA formed by a set of invariant residues (site I: Cys382, Cys384, and Tyr688; site II: Tyr688, Asn689, Met717, and Ser721) (16).…”

“…For instance, a range of mutations in the human Cu þ -transporting class IB P-type (P IB-1 ) ATPases ATP7A and ATP7B give rise to the severe Menkes' and Wilson's diseases, which manifest as copper deficiency and overload, respectively (8). The recent crystal structures of a bacterial homolog from Legionella pneumophila (LpCopA) (9,10) and the closely related Zn 2þ -transporting P IB-2 -ATPases (11) have provided a structural framework for elucidating transition-metal P-type (P IB ) ATPase transport and related diseases. However, the Cu þ -and Zn 2þ -ATPase crystal structures and conserved sequence motifs show significant differences in the intramembranous ion-binding sites (12)(13)(14)(15)(16), which suggests that the ion-entry mechanisms and determinants of ion specificity are likely unique to each P-type ATPase heavy-metal transporter.…”