Multiple Metal Binding Domains Enhance the Zn(II) Selectivity of the Divalent Metal Ion Transporter AztA

Complex metal homeostasis and trafficking systems control the bioavailability of essential transition metal ions while ensuring that these and other abiological xenobiotics, including Cd, Pb, Hg, and As, do not accumulate inside cells. The heavy metal ion-transporting CPx-ATPases represent a large subfamily of P-type ATPases (P1B-type) found in both prokaryotes and eukaryotes that play important roles in metal homeostasis (1–5). All are characterized by eight transmembrane (TM)1 helices that likely form the channel for transport. TM6 bears the CPx signature sequence, which is thought, in conjunction with other residues in the membrane helices, to coordinate the metal during transport (6). Large cytoplasmic loops are folded into structurally characterized actuator (7) [A-domain (Figure 1A)] and ATP binding domains (8, 9) (N- and P-domains), with metal translocation coupled to phosphorylation of an aspartate residue in the P-domain. Figure 1 (A) Schematic representation of the domain structure of AztA showing the two tandem MBDs followed by an ≈30-residue hydrophilic His-rich linker. The predicted eight transmembrane helices, cytosolic ATP binding domains (N and P), and actuator domain ... Another significant feature of CPx-ATPases is that most have N-terminal and/or C-terminal cytosolic extensions, often containing one or more tandemly linked ferredoxin fold-like βαββαβ metal binding domains (MBDs) (Figure 1A) (10). For example, the Wilson's and Menkes disease Cu/Ag-specific ATPases ATP7A and ATP7B, respectively, have six tandemly linked MBDs, while those from lower eukaryotes and most prokaryotes have zero, one, or two MBDs. These MBDs are known to provide docking sites for Cu chaperones that allow Cu to be handed off, via intermolecular metal–ligand exchange reactions, to partner MBDs without dissociation of the metal into bulk solution (11–14). This provides strong support for the central tenet of the Cu-trafficking hypothesis (15, 16). Mechanistic studies with Archaeglobus fulgidis CopA suggest that metal binding to the single N-terminal MBD plays a regulatory role in enhancing the rate of dephosphorylation of the phosphoaspartate residue in the E2 state; this increases the rate of metal ion release which is rate-limiting in multiple-turnover experiments (17). Some divalent metal ion (Zn/Cd/Pb)-specific P1B-type ATPases (2) are also known to possess an MBD, but the functional role that this domain plays is a topic of ongoing investigation. Since there are no known zinc chaperones, the significance of protein–protein docking and intermolecular transfer is unclear. However, there is some evidence in support of the idea that the specific structural features of individual MBDs might provide some metal selectivity to the transporter itself, despite the fact that they all adopt essentially the same βαββαβ fold (10) and all metal complexes employ the two conserved Cys residues of the CXXC sequence as metal donor atoms (18–21). For example, in both copper chaperones and MBDs derived from Cu/Ag transporters, the Cu(I) is often coordinated via a linear bis-thiolate complex (22–24); in another case, a distorted trigonal S2N complex is found, where a His derived from loop 5 between helix α2 and strand β4 is a ligand (25). For the Zn/Cd/Pb transporter Escherichia coli ZntA, a conserved Asp just N-terminal to the first Cys (DCXXC) was proposed to drive 3- or 4-coordination of Zn(II) (19); in contrast, for the Cd/Pb-selective transporter Listeria monocytogenes CadA, a conserved Glu in loop 5 (E61 in Figure 1B) appears to form a coordination bond to the Cd(II) in a binuclear homodimeric subunit bridging structure (20). Anabaena AztA (alr7622) is a P1B-type ATPase efflux pump encoded by the azt (Anabaena zinc transport) operon whose expression is transcriptionally induced upon direct binding of Zn, Pb, or Cd by the ArsR (or ArsR/SmtB) family (26) regulator AztR (27). AztA possesses the unique functional property of conferring significant Zn(II) resistance to a transformed Zn/Cd/Pb-hypersensitive E. coli strain (GG48), relative to Cd(II) and Pb(II) (27). In fact, cadmium and lead resistance is barely detectable relative to that conferred by other known Pb/Cd/Zn-transporting ATPases, including E. coli ZntA (28) and CadAs from Streptomyces aureus, Ralstonia metallodurans, and L. monocytogenes (29). AztA is also distinguished from other characterized Zn/Cd/ Pb transporters on the basis of harboring two N-terminal MBDs, each of which is followed by an ≈30-residue hydrophilic His-rich linker (Figure 1). The two MBDs of AztA are 44% identical, pairwise, in amino acid sequence (71% similar), and each contains a DCXXC sequence and lacks the Glu in loop 5 found in CadAs. Since AztA is unique in incorporating tandem MBDs followed by His-rich segments, this led us to hypothesize that some aspect of these specific features might confer higher selectivity for Zn(II) relative to Cd(II) and Pb(II). In this report, we show that the two MBDs of Anabaena AztA play nonredundant structural and functional roles in metal binding and heavy metal resistance in vivo, but only with respect to zinc resistance. We show that the membrane proximal b-MBD has a higher affinity for Zn(II) than the distal a-MBD, and consistent with this, Zn(II) prebound to the a-MBD facilely moves to a metal-free b-MBD. However, the low-affinity a-MBD plays a critical role in mediating zinc resistance under conditions of high zinc toxicity in a manner that does not require the b-MBD. This suggests that the a-MBD is capable of functioning in a manner independent of the b-MBD in maximally stimulating transport under high intracellular zinc loads. In contrast, both Cd(II) and Pb(II) form stable 1:1 S4 or S3(N/O) complexes in AztAaHbH, where a single metal ion bridges the two Cys-X2-Cys sites from aand b-MBDs or forms intermolecular a–a′ and b–b′ cross-linked structures. We hypothesize that these structures represent kinetically trapped intermediates that are poor substrates or regulators for metal transport by AztA.