Cytoplasmic Metal-binding Domains Research Articles

Characterization of the substrate binding site of an iron detoxifying membrane transporter from Plasmodium falciparum

BackgroundPlasmodium species are entirely dependent upon their host as a source of essential iron. Although it is an indispensable micronutrient, oxidation of excess ferrous iron to the ferric state in the cell cytoplasm can produce reactive oxygen species that are cytotoxic. The malaria parasite must therefore carefully regulate the processes involved in iron acquisition and storage. A 273 amino acid membrane transporter that is a member of the vacuolar iron transporter (VIT) family and an orthologue of the yeast Ca2+-sensitive cross complementer (CCC1) protein plays a major role in cytosolic iron detoxification of Plasmodium species and functions in transport of ferrous iron ions into the endoplasmic reticulum for storage. While this transporter, termed PfVIT, is not critical for viability of the parasite evidence from studies of mice infected with VIT-deficient Plasmodium suggests it could still provide an efficient target for chemoprophylactic treatment of malaria. Individual amino acid residues that constitute the Fe2+ binding site of the protein were identified to better understand the structural basis of substrate recognition and binding by PfVIT.MethodsUsing the crystal structure of a recently published plant VIT as a template, a high-quality homology model of PfVIT was constructed to identify the amino acid composition of the transporter’s substrate binding site and to act as a guide for subsequent mutagenesis studies. To test the effect of mutation of the substrate binding-site residues on PfVIT function a yeast complementation assay assessed the ability of overexpressed, recombinant wild type and mutant PfVIT to rescue an iron-sensitive deletion strain (ccc1∆) of Saccharomyces cerevisiae yeast from the toxic effects of a high concentration of extracellular iron.ResultsThe combined in silico and mutagenesis approach identified a methionine residue located within the cytoplasmic metal binding domain of the transporter as essential for PfVIT function and provided insight into the structural basis for the Fe2+-selectivity of the protein.ConclusionThe structural model of the metal binding site of PfVIT opens the door for rational design of therapeutics to interfere with iron homeostasis within the malaria parasite.

Copper relay path through the N-terminus of Wilson disease protein, ATP7B

In human cells, copper (Cu) ions are transported by the cytoplasmic Cu chaperone Atox1 to the Wilson disease protein (ATP7B) in the Golgi for loading of Cu-dependent enzymes. ATP7B is a membrane-spanning protein which, in contrast to non-mammalian homologs, has six cytoplasmic metal-binding domains (MBDs). To address the reason for multiple MBDs, we introduced strategic mutations in which one, two or three MBDs had been blocked for Cu binding via cysteine-to-serine mutations (but all six MBDs are present in all) in a yeast system that probes Cu flow through Atox1 and ATP7B. The results, combined with earlier work, support a mechanistic model in which MBD1-3 forms a regulatory unit of ATP7B Cu transport. Cu delivery via Atox1 to this unit, followed by loading of Cu in MBD3, promotes release of inhibitory interactions. Whereas the Cu site in MBD4 can be mutated without a large effect, an intact Cu site in either MBD5 or MBD6 is required for Cu transport. All MBDs, expressed as single-domain proteins, can replace Atox1 and deliver Cu to full-length ATP7B. However, only MBD6 can deliver Cu to truncated ATP7B where all six MBDs are removed, suggesting a docking role for this structural unit.

Disease-causing point-mutations in metal-binding domains of Wilson disease protein decrease stability and increase structural dynamics

After cellular uptake, Copper (Cu) ions are transferred from the chaperone Atox1 to the Wilson disease protein (ATP7B) for incorporation into Cu-dependent enzymes in the secretory pathway. Human ATP7B is a large multi-domain membrane-spanning protein which, in contrast to homologues in other organisms, has six similar cytoplasmic metal-binding domains (MBDs). The reason for multiple MBDs is proposed to be indirect modulation of enzymatic activity and it is thus intriguing that point mutations in MBDs can promote Wilson disease. We here investigated, in vitro and in silico, the biophysical consequences of clinically-observed Wilson disease mutations, G85V in MBD1 and G591D in MBD6, incorporated in domain 4. Because G85 and G591 correspond to a conserved Gly found in all MBDs, we introduced the mutations in the well-characterized MBD4. We found the mutations to dramatically reduce the MBD4 thermal stability, shifting the midpoint temperature of unfolding by more than 20 °C. In contrast to wild type MBD4 and MBD4D, MBD4V adopted a misfolded structure with a large β-sheet content at high temperatures. Molecular dynamic simulations demonstrated that the mutations increased backbone fluctuations that extended throughout the domain. Our findings imply that reduced stability and enhanced dynamics of MBD1 or MBD6 is the origin of ATP7B dysfunction in Wilson disease patients with the G85V or G591D mutation.

Metal binding to the N-terminal cytoplasmic domain of the PIB ATPase HMA4 is required for metal transport in Arabidopsis.

PIB ATPases are metal cation pumps that transport metals across membranes. These proteins possess N- and C-terminal cytoplasmic extensions that contain Cys- and His-rich high affinity metal binding domains, which may be involved in metal sensing, metal ion selectivity and/or in regulation of the pump activity. The PIB ATPase HMA4 (Heavy Metal ATPase 4) plays a central role in metal homeostasis in Arabidopsis thaliana and has a key function in zinc and cadmium hypertolerance and hyperaccumulation in the extremophile plant species Arabidopsis halleri. Here, we examined the function and structure of the N-terminal cytoplasmic metal-binding domain of HMA4. We mutagenized a conserved CCTSE metal-binding motif in the domain and assessed the impact of the mutations on protein function and localization in planta, on metal-binding properties in vitro and on protein structure by Nuclear Magnetic Resonance spectroscopy. The two Cys residues of the motif are essential for the function, but not for localization, of HMA4 in planta, whereas the Glu residue is important but not essential. These residues also determine zinc coordination and affinity. Zinc binding to the N-terminal domain is thus crucial for HMA4 protein function, whereas it is not required to maintain the protein structure. Altogether, combining in vivo and in vitro approaches in our study provides insights towards the molecular understanding of metal transport and specificity of metal P-type ATPases.

Enthalpy-entropy compensation at play in human copper ion transfer.

Copper (Cu) is an essential trace element but toxic in free form. After cell uptake, Cu is transferred, via direct protein-protein interactions, from the chaperone Atox1 to the Wilson disease protein (WD) for incorporation into Cu-dependent enzymes. Cu binds to a conserved C1XXC2 motif in the chaperone as well as in each of the cytoplasmic metal-binding domains of WD. Here, we dissect mechanism and thermodynamics of Cu transfer from Atox1 to the fourth metal binding domain of WD. Using chromatography and calorimetry together with single Cys-to-Ala variants, we demonstrate that Cu-dependent protein heterocomplexes require the presence of C1 but not C2. Comparison of thermodynamic parameters for mutant versus wild type reactions reveals that the wild type reaction involves strong entropy-enthalpy compensation. This property is explained by a dynamic inter-conversion of Cu-Cys coordinations in the wild type ensemble and may provide functional advantage by protecting against Cu mis-ligation and bypassing enthalpic traps.

Bacterial Transition Metal P1B-ATPases: Transport Mechanism and Roles in Virulence

P(1B)-type ATPases are polytopic membrane proteins that couple the hydrolysis of ATP to the efflux of cytoplasmic transition metals. This paper reviews recent progress in our understanding of the structure and function of these proteins in bacteria. These are members of the P-type superfamily of transport ATPases. Cu(+)-ATPases are the most frequently observed and best-characterized members of this group of transporters. However, bacterial genomes show diverse arrays of P(1B)-type ATPases with a range of substrates (Cu(+), Zn(2+), Co(2+)). Furthermore, because of the structural similarities among transitions metals, these proteins can also transport nonphysiological substrates (Cd(2+), Pb(2+), Au(+), Ag(+)). P(1B)-type ATPases have six or eight transmembrane segments (TM) with metal coordinating amino acids in three core TMs flanking the cytoplasmic domain responsible for ATP binding and hydrolysis. In addition, regulatory cytoplasmic metal binding domains are present in most P(1B)-type ATPases. Central to the transport mechanism is the binding of the uncomplexed metal to these proteins when cytoplasmic substrates are bound to chaperone and chelating molecules. Metal binding to regulatory sites is through a reversible metal exchange among chaperones and cytoplasmic metal binding domains. In contrast, the chaperone-mediated metal delivery to transport sites appears as a largely irreversible event. P(1B)-ATPases have two overarching physiological functions: to maintain cytoplasmic metal levels and to provide metals for the periplasmic assembly of metalloproteins. Recent studies have shown that both roles are critical for bacterial virulence, since P(1B)-ATPases appear key to overcome high phagosomal metal levels and are required for the assembly of periplasmic and secreted metalloproteins that are essential for survival in extreme oxidant environments.

Chaperone-mediated Cu+ Delivery to Cu+ Transport ATPases: REQUIREMENT OF NUCLEOTIDE BINDING

Cu(+)-ATPases drive the efflux of Cu(+) from the cell cytoplasm. During their catalytic/transport cycle, cytoplasmic Cu(+)-chaperones deliver the metal to the two transmembrane metal-binding sites (TM-MBSs) responsible for Cu(+) translocation. Here, using Archaeoglobus fulgidus Cu(+)-ATPase CopA and the C-terminal Cu(+)-chaperone domain of CopZ (Ct-CopZ), we describe the mechanism of Cu(+) transfer to both TM-MBSs. In absence of other ligands, Ct-CopZ transfers Cu(+) to wild-type CopA and to various CopA constructs lacking or having mutated cytoplasmic metal-binding domains, in a fashion consistent with occupancy of a single TM-MBS. Similar experiments performed in the presence of 2.5 mm ADP-Mg(2+), stabilizing an E1.ADP, lead to full occupancy of both TM-MBSs. In both cases, the transfer is largely stoichiometric, i.e. equimolar amounts of Ct-CopZ.Cu(+) saturated the TM-MBSs. Experiments performed with CopA mutants lacking either TM-MBS showed that both sites are loaded independently, and nucleotide binding does not affect their availability. The nucleotide-induced E2-->E1 transition is structurally characterized by a large displacement of the A and N domains opening the cytoplasmic region of P-type ATPases. Then, it is apparent that, whereas the first Cu(+)-chaperone can bind an ATPase form available in the absence of ligands, the second requires the E1.nucleotide intermediary to interact and deliver the metal. Interestingly, independent of TM-MBS Cu(+) loading, nucleotide binding also prevents the regulatory interaction of the N-terminal cytoplasmic metal-binding domain with the nucleotide binding domain.

Mechanism of Cu+-transporting ATPases: Soluble Cu+chaperones directly transfer Cu+to transmembrane transport sites

As in other P-type ATPases, metal binding to transmembrane metal-binding sites (TM-MBS) in Cu(+)-ATPases is required for enzyme phosphorylation and subsequent transport. However, Cu(+) does not access Cu(+)-ATPases in a free (hydrated) form but is bound to a chaperone protein. Cu(+) transfer from Cu(+) chaperones to regulatory cytoplasmic metal-binding domains (MBDs) present in these ATPases has been described, but there is no evidence of a proposed subsequent Cu(+) movement from the MBDs to the TM-MBS. Alternatively, we postulate the parsimonious Cu(+) transfer by the chaperone directly to TM-MBS. Testing both models, the delivery of Cu(+) by Archaeoglobus fulgidus Cu(+) chaperone CopZ to the corresponding Cu(+)-ATPase, CopA, was studied. As expected, CopZ interacted with and delivered the metal to CopA MBDs. Cu(+)-loaded MBDs, acting as metal donors, were unable to activate CopA or a truncated CopA lacking MBDs. Conversely, Cu(+)-loaded CopZ activated the CopA ATPase and CopA constructs in which MBDs were rendered unable to bind Cu(+). Furthermore, under nonturnover conditions, CopZ transferred Cu(+) to the TM-MBS of a CopA lacking MBDs. These data are consistent with a model where MBDs serve a regulatory function without participating in metal transport and the chaperone delivers Cu(+) directly to transmembrane transport sites of Cu(+)-ATPases.

The structure and function of heavy metal transport P1B-ATPases

P(1B)-type ATPases transport heavy metals (Cu+, Cu2+, Zn2+, Co2+, Cd2+, Pb2+) across membranes. Present in most organisms, they are key elements for metal homeostasis. P(1B)-type ATPases contain 6-8 transmembrane fragments carrying signature sequences in segments flanking the large ATP binding cytoplasmic loop. These sequences made possible the differentiation of at least four P(1B)-ATPase subgroups with distinct metal selectivity: P(1B-1): Cu+, P(1B-2): Zn2+, P(1B-3): Cu2+, P(1B-4): Co2+. Mutagenesis of the invariant transmembrane Cys in H6, Asn and Tyr in H7 and Met and Ser in H8 of the Archaeoglobus fulgidus Cu+-ATPase has revealed that their side chains likely coordinate the metals during transport and constitute a central unique component of these enzymes. The structure of various cytoplasmic domains has been solved. The overall structure of those involved in enzyme phosphorylation (P-domain), nucleotide binding (N-domain) and energy transduction (A-domain), appears similar to those described for the SERCA Ca2+-ATPase. However, they show different features likely associated with singular functions of these proteins. Many P(1B)-type ATPases, but not all of them, also contain a diverse arrangement of cytoplasmic metal binding domains (MBDs). In spite of their structural differences, all N- and C-terminal MBDs appear to control the enzyme turnover rate without affecting metal binding to transmembrane transport sites. In addition, eukaryotic Cu+-ATPases have multiple N-MBD regions that participate in the metal dependent targeting and localization of these proteins. The current knowledge of structure-function relationships among the different P(1B)-ATPases allows for a description of selectivity, regulation and transport mechanisms. Moreover, it provides a framework to understand mutations in human Cu+-ATPases (ATP7A and ATP7B) that lead to Menkes and Wilson diseases.

Structure of the ATP Binding Domain from the Archaeoglobus fulgidus Cu+-ATPase

The P-type ATPases translocate cations across membranes using the energy provided by ATP hydrolysis. CopA from Archaeoglobus fulgidus is a hyperthermophilic ATPase responsible for the cellular export of Cu+ and is a member of the heavy metal P1B-type ATPase subfamily, which includes the related Wilson and Menkes diseases proteins. The Cu+-ATPases are distinct from their P-type counter-parts in ion binding sequences, membrane topology, and the presence of cytoplasmic metal binding domains, suggesting that they employ alternate forms of regulation and novel mechanisms of ion transport. To gain insight into Cu+-ATPase function, the structure of the CopA ATP binding domain (ATPBD) was determined to 2.3 A resolution. Similar to other P-type ATPases, the ATPBD includes nucleotide binding (N-domain) and phosphorylation (P-domain) domains. The ATPBD adopts a closed conformation similar to the nucleotide-bound forms of the Ca2+-ATPase. The CopA ATPBD is much smaller and more compact, however, revealing the minimal elements required for ATP binding, hydrolysis, and enzyme phosphorylation. Structural comparisons to the AMP-PMP-bound form of the Escherichia coli K+-transporting Kdp-ATPase and to the Wilson disease protein N-domain indicate that the five conserved N-domain residues found in P1B-type ATPases, but not in the other families, most likely participate in ATP binding. By contrast, the P-domain includes several residues conserved among all P-type ATPases. Finally, the CopA ATPBD structure provides a basis for understanding the likely structural and functional effects of various mutations that lead to Wilson and Menkes diseases.

Identification of the Transmembrane Metal Binding Site in Cu+-transporting PIB-type ATPases

P(IB)-type ATPases have an essential role maintaining copper homeostasis. Metal transport by these membrane proteins requires the presence of a transmembrane metal occlusion/binding site. Previous studies showed that Cys residues in the H6 transmembrane segment are required for metal transport. In this study, the participation in metal binding of conserved residues located in transmembrane segments H7 and H8 was tested using CopA, a model Cu(+)-ATPase from Archaeoglobus fulgidus. Four invariant amino acids in the central portion of H7 (Tyr(682) and Asn(683)) and H8 (Met(711) and Ser(715)) were identified as required for Cu(+) binding. Replacement of these residues abolished enzyme activity. These proteins did not undergo Cu(+)-dependent phosphorylation by ATP but were phosphorylated by P(i) in the absence of Cu(+). Moreover, the presence of Cu(+) could not prevent the enzyme phosphorylation by P(i). Other conserved residues in the H7-H8 region were not required for metal binding. Mutation of two invariant Pro residues had little effect on enzyme function. Replacement of residues located close to the cytoplasmic end of H7-H8 led to inactive enzymes. However, these were able to interact with Cu(+) and undergo phosphorylation. This suggests that the integrity of this region is necessary for conformational transitions but not for ligand binding. These data support the presence of a unique transmembrane Cu(+) binding/translocation site constituted by Tyr-Asn in H7, Met and Ser in H8, and two Cys in H6 of Cu(+)-ATPases. The likely Cu(+) coordination during transport appears distinct from that observed in Cu(+) chaperone proteins or catalytic/redox metal binding sites.

Cd2+ and the N-terminal metal-binding domain protect the putative membranous CPC motif of the Cd2+-ATPase of Listeria monocytogenes

CadA, the Cd(2+)-ATPase of Listeria monocytogenes, contains four cysteine residues: two in the CTNC (Cys-Thr-Asn-Cys) sequence in the cytoplasmic metal-binding domain (MBD), and two in the CPC (Cys-Pro-Cys) sequence in the membrane domain. Taking advantage of DeltaMBD, a truncated version of CadA that lacks the MBD but which still acts as a functional Cd(2+)-ATPase [Bal, Mintz, Guillain and Catty (2001) FEBS Lett. 506, 249-252], we analysed the role of the membrane cysteine residues (studied using DeltaMBD) separately from that of the cysteine residues of the MBD, which were studied using full-length CadA. The role of the cysteines was assessed by reacting DeltaMBD and CadA with N -ethylmaleimide (NEM), an SH-specific reagent, in the presence or absence of Cd(2+). We show here that (i) in both DeltaMBD and CadA, the cysteine residues in the CPC motif are essential for phosphorylation; (ii) in both proteins, Cd(2+) protects against alkylation by NEM; and (iii) in the absence of Cd(2+), the MBD of CadA also protects against alkylation by NEM. Our results suggest that the CPC motif is present in the membrane Cd(2+) transport site(s) and that the MBD protects these site(s).