Structure of the Two Transmembrane Cu+ Transport Sites of the Cu+-ATPases

Abstract

Cu؉ -ATPases drive metal efflux from the cell cytoplasm. Paramount to this function is the binding of Cu ؉ within the transmembrane region and its coupled translocation across the permeability barrier. Copper is an essential micronutrient (1, 2). It has critical catalytic and electron transfer roles in a number of key proteins (tyrosinase, lysyl oxidase, ferroxidase ceruloplasmin, plastocyanin, etc.). However, when free, copper participates in the production of reactive oxygen species leading to cellular damage. Toward sustaining intracellular copper balance, transmembrane transport systems maintain the copper cell quota, Cu ϩ chaperone proteins traffic the bound metal to specific cellular targets, and metal-sensing transcription factors control copper dependent protein expression (3-5). The metal coordination geometry in these proteins is central to the efficiency of the Cu ϩ mobilization processes. In this direction, the coordination should ensure the specificity and prevent the release of free Cu ϩ to the cytoplasm. Canonical copper metalloproteins have long been characterized and classified based on spectroscopic and magnetic properties (Types I, II, and III) (6 -8). Their study has provided great detail on copper coordination in "permanent" sites where copper is bound during the functional life of the proteins. Cu ϩ linear coordination by invariant Cys residues of chaperone proteins has been described, providing insight into the mechanism of copper trafficking and exchange among similar domains (9, 10). More recently, trigonal coordination by Cys 2 -His sites has been observed, for instance, in Mycobacterium tuberculosis transcription factor CsoR (11). Alternatively, Met n His was found in several Cu ϩ -trafficking proteins located in the oxidizing periplasm of prokaryotes (12)(13)(14). Despite this progress, Cu ϩ distribution and balance cannot be understood without describing the selective coordination during compartmental transmembrane transport.In eukaryotic cells, members of the Ctr family of proteins transport Cu ϩ inside the cell (15). Ctr1 organizes as homotrimers forming transmembrane pores that facilitate Cu ϩ transmembrane translocation by an apparently energy-independent undefined mechanism (16, 17). Although relevant Cu ϩ -binding Met have been observed in the extracellular loops of Ctr1 (15); none of the invariant transmembrane residues appear to be required for transport, and no direct coordination is evident (17).As a counterpart to influx systems, Cu ϩ -ATPases are responsible for cytoplasmic Cu ϩ efflux. Mutations of the human Cu ϩ -ATPase genes, ATP7A and ATP7B, lead to Menkes syndrome and Wilson disease, respectively (18,19). Cu ϩ -ATPases are members of the superfamily of P-type ATPases (20, 21). These couple Cu ϩ transport to the hydrolysis of ATP, following a classical Post catalytic/transport cycle (19,21). In this mechanism, transmembrane metal-binding sites (TMMBSs) 4 are responsible for handling the ion during transmembrane translocation (22). These transmembrane sites are expos...

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

“…5 C). We observed that the ion came to a halt when it encountered Met717 (referred to as site III), which correlates with its assigned role as an internal ion-binding residue (16). The faster pull rate reproduced these overall results (Fig.…”

“…During equilibration of the LpCopA E1 model, water molecules entered into a cytoplasmic cavity that extended toward internal amino acid residues that have been proposed to participate in ion binding (14,16), which is in accordance with an E1 state exposing its internal binding site to the cytoplasm (Fig. S9 A).…”

“…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). Similarly, a two-ion stoichiometry per protein was observed in plant Cu þ ATPases (28).…”

“…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. Here, we address the central question of how the transported Cu þ ion is delivered to the protein interior for further ATPdependent transport.…”

“…Here, E1 and E2P denote states in which the TM domains are open toward the cytoplasm and noncytoplasmic space, respectively. Conformational changes associated with autophosphorylation (from E1 to E1P) and autodephosphorylation (E2P to E2) in the A-, P-, and N-domains are coupled with changes in accessibility to intramembranous ion-binding residues in the TM portion of the protein, which results in ion transport (16)…”