Structure and interactions of the C‐terminal metal binding domain of Archaeoglobus fulgidus CopA

The Cu⁺‐ATPase CopA from Archaeoglobus fulgidus belongs to the P_1B family of the P‐type ATPases. These integral membrane proteins couple the energy of ATP hydrolysis to heavy metal ion translocation across membranes. A defining feature of P_1B‐1‐type ATPases is the presence of soluble metal binding domains at the N‐terminus (N‐MBDs). The N‐MBDs exhibit a conserved ferredoxin‐like fold, similar to that of soluble copper chaperones, and bind metal ions via a conserved CXXC motif. The N‐MBDs enable Cu⁺ regulation of turnover rates apparently through Cu‐sensitive interactions with catalytic domains. A. fulgidus CopA is unusual in that it contains both an N‐terminal MBD and a C‐terminal MBD (C‐MBD). The functional role of the unique C‐MBD has not been established. Here, we report the crystal structure of the apo, oxidized C‐MBD to 2.0 Å resolution. In the structure, two C‐MBD monomers form a domain‐swapped dimer, which has not been observed previously for similar domains. In addition, the interaction of the C‐MBD with the other cytoplasmic domains of CopA, the ATP binding domain (ATPBD) and actuator domain (A‐domain), has been investigated. Interestingly, the C‐MBD interacts specifically with both of these domains, independent of the presence of Cu⁺ or nucleotides. These data reinforce the uniqueness of the C‐MBD and suggest a distinct structural role for the C‐MBD in CopA transport. Proteins 2010. © 2010 Wiley‐Liss, Inc.     

Distinct functions of serial metal‐binding domains in the Escherichia coli P1B‐ATPase CopA

P_1B‐ATPases are among the most common resistance factors to metal‐induced stress. Belonging to the superfamily of P‐type ATPases, they are capable of exporting transition metal ions at the expense of adenosine triphosphate (ATP) hydrolysis. P_1B‐ATPases share a conserved structure of three cytoplasmic domains linked by a transmembrane domain. In addition, they possess a unique class of domains located at the N‐terminus. In bacteria, these domains are primarily associated with metal binding and either occur individually or as serial copies of each other. Within this study, the roles of the two adjacent metal‐binding domains (MBDs) of CopA, the copper export ATPase of Escherichia coli were investigated. From biochemical and physiological data, we deciphered the protein‐internal pathway of copper and demonstrate the distal N‐terminal MBD to possess a function analogous to the metallochaperones of related prokaryotic copper resistance systems, that is its involvement in the copper transfer to the membrane‐integral ion‐binding sites of CopA. In contrast, the proximal domain MBD2 has a regulatory role by suppressing the catalytic activity of CopA in absence of copper. Furthermore, we propose a general functional divergence of tandem MBDs in P_1B‐ATPases, which is governed by the length of the inter‐domain linker.     

The transport mechanism of bacterial Cu<Superscript>+</Superscript>-ATPases: distinct efflux rates adapted to different function

Cu⁺-ATPases play a key role in bacterial Cu⁺ homeostasis by participating in Cu⁺ detoxification and cuproprotein assembly. Characterization of Archaeoglobus fulgidus CopA, a model protein within the subfamily of P_1B-1 type ATPases, has provided structural and mechanistic details on this group of transporters. Atomic resolution structures of cytoplasmic regulatory metal binding domains (MBDs) and catalytic actuator, phosphorylation, and nucleotide binding domains are available. These, in combination with whole protein structures resulting from cryo-electron microscopy analyses, have enabled the initial modeling of these transporters. Invariant residues in helixes 6, 7 and 8 form two transmembrane metal binding sites (TM-MBSs). These bind Cu⁺ with high affinity in a trigonal planar geometry. The cytoplasmic Cu⁺ chaperone CopZ transfers the metal directly to the TM-MBSs; however, loading both of the TM-MBSs requires binding of nucleotides to the enzyme. In agreement with the classical transport mechanism of P-type ATPases, occupancy of both transmembrane sites by cytoplasmic Cu⁺ is a requirement for enzyme phosphorylation and subsequent transport into the periplasmic or extracellular milieus. Recent transport studies have shown that all Cu⁺-ATPases drive cytoplasmic Cu⁺ efflux, albeit with quite different transport rates in tune with their various physiological roles. Archetypical Cu⁺-efflux pumps responsible for Cu⁺ tolerance, like the Escherichia coli CopA, have turnover rates ten times higher than those involved in cuproprotein assembly (or alternative functions). This explains the incapability of the latter group to significantly contribute to the metal efflux required for survival in high copper environments.     

Walker‐A threonine couples nucleotide occupancy with the chaperone activity of the AAA+ ATPase ClpB

Hexameric AAA+ ATPases induce conformational changes in a variety of macromolecules. AAA+ structures contain the nucleotide‐binding P‐loop with the Walker A sequence motif: GxxGxGK(T/S). A subfamily of AAA+ sequences contains Asn in the Walker A motif instead of Thr or Ser. This noncanonical subfamily includes torsinA, an ER protein linked to human dystonia and DnaC, a bacterial helicase loader. Role of the noncanonical Walker A motif in the functionality of AAA+ ATPases has not been explored yet. To determine functional effects of introduction of Asn into the Walker A sequence, we replaced the Walker‐A Thr with Asn in ClpB, a bacterial AAA+ chaperone which reactivates aggregated proteins. We found that the T‐to‐N mutation in Walker A partially inhibited the ATPase activity of ClpB, but did not affect the ClpB capability to associate into hexamers. Interestingly, the noncanonical Walker A sequence in ClpB induced preferential binding of ADP vs. ATP and uncoupled the linkage between the ATP‐bound conformation and the high‐affinity binding to protein aggregates. As a consequence, ClpB with the noncanonical Walker A sequence showed a low chaperone activity in vitro and in vivo. Our results demonstrate a novel role of the Walker‐A Thr in sensing the nucleotide's γ‐phosphate and in maintaining an allosteric linkage between the P‐loop and the aggregate binding site of ClpB. We postulate that AAA+ ATPases with the noncanonical Walker A might utilize distinct mechanisms to couple the ATPase cycle with their substrate‐remodeling activity.     

Properties of the P-Type ATPases Encoded by the copAP Operons of Helicobacter pylori and Helicobacter felis

The cop operons of Helicobacter pylori and Helicobacter felis were cloned by gene library screening. Both operons contain open reading frames for a P-type ion pump (CopA) with homology to Cd²⁺ and Cu²⁺ ATPases and a putative ion binding protein (CopP), the latter representing a CopZ homolog of the copYZAB operon of Enterococcus hirae. The predicted CopA ATPases contained an N-terminal GMXCXXC ion binding motif and a membrane-associated CPC sequence. A synthetic N-terminal peptide of the H. pylori CopA ATPase bound to Cu²⁺ specifically, and gene disruption mutagenesis of CopA resulted in an enhanced growth sensitivity of H. pylori to Cu²⁺ but not to other divalent cations. As determined experimentally, H. pylori CopA contains four pairs of transmembrane segments (H1 to H8), with the ATP binding and phosphorylation domains lying between H6 and H7, as found for another putative transition metal pump of H. pylori (K. Melchers, T. Weitzenegger, A. Buhmann, W. Steinhilber, G. Sachs, and K. P. Schäfer, J. Biol. Chem. 271:446–457, 1996). The corresponding transmembrane segments of the H. felis CopA pump were identified by hydrophobicity analysis and via sequence similarity. To define functional domains, similarly oriented regions of the two enzymes were examined for sequence identity. Regions with high degrees of identity included the N-terminal Cu²⁺ binding domain, the regions of ATP binding and phosphorylation in the energy transduction domain, and a transport domain consisting of the last six transmembrane segments with conserved cysteines in H4, H6, and H7. The data suggest that H. pylori and H. felis employ conserved mechanisms of ATPase-dependent copper resistance.     

Activation of Archaeoglobus fulgidus Cu-ATPase CopA by cysteine

CopA, a thermophilic ATPase from Archaeoglobus fulgidus, drives the outward movement of Cu⁺ across the cell membrane. Millimolar concentration of Cys dramatically increases (≅ 800%) the activity of CopA and other P_IB-type ATPases (Escherichia coli ZntA and Arabidopsis thaliana HMA2). The high affinity of CopA for metal (≅ 1 μM) together with the low Cu⁺-Cys K_D (< 10^− 10M) suggested a multifaceted interaction of Cys with CopA, perhaps acting as a substitute for the Cu⁺ chaperone protein present in vivo. To explain the activation by the amino acid and further understand the mechanism of metal delivery to transport ATPases, Cys effects on the turnover and partial reactions of CopA were studied. 2-20 mM Cys accelerates enzyme turnover with little effect on CopA affinity for Cu⁺, suggesting a metal independent activation. Furthermore, Cys activates the p-nitrophenyl phosphatase activity of CopA, even though this activity is metal independent. Cys accelerates enzyme phosphorylation and the forward dephosphorylation rates yielding higher steady state phosphoenzyme levels. The faster dephosphorylation would explain the higher enzyme turnover in the presence of Cys. The amino acid has no significant effect on low affinity ATP K_m suggesting no changes in the E₁ ↔ E₂ equilibrium. Characterization of Cu⁺ transport into sealed vesicles indicates that Cys acts on the cytoplasmic side of the enzyme. However, the Cys activation of truncated CopA lacking the N-terminal metal binding domain (N-MBD) indicates that activation by Cys is independent of the regulatory N-MBD. These results suggest that Cys is a non-essential activator of CopA, interacting with the cytoplasmic side of the enzyme while this is in an E1 form. Interestingly, these effects also point out that Cu⁺ can reach the cytoplasmic opening of the access path into the transmembrane transport sites either as a free metal or a Cu⁺-Cys complex.     

Structural analysis of ligand‐bound states of the Salmonella type III secretion system ATPase InvC

Translocation of virulence effector proteins through the type III secretion system (T3SS) is essential for the virulence of many medically relevant Gram‐negative bacteria. The T3SS ATPases are conserved components that specifically recognize chaperone–effector complexes and energize effector secretion through the system. It is thought that functional T3SS ATPases assemble into a cylindrical structure maintained by their N‐terminal domains. Using size‐exclusion chromatography coupled to multi‐angle light scattering and native mass spectrometry, we show that in the absence of the N‐terminal oligomerization domain the Salmonella T3SS ATPase InvC can form monomers and dimers in solution. We also present for the first time a 2.05 å resolution crystal structure of InvC lacking the oligomerization domain (InvCΔ79) and map the amino acids suggested for ATPase intersubunit interaction, binding to other T3SS proteins and chaperone–effector recognition. Furthermore, we validate the InvC ATP‐binding site by co‐crystallization of InvCΔ79 with ATPγS (2.65 å) and ADP (2.80 å). Upon ATP‐analogue recognition, these structures reveal remodeling of the ATP‐binding site and conformational changes of two loops located outside of the catalytic site. Both loops face the central pore of the predicted InvC cylinder and are essential for the function of the T3SS ATPase. Our results present a fine functional and structural correlation of InvC and provide further details of the homo‐oligomerization process and ATP‐dependent conformational changes underlying the T3SS ATPase activity.     

Molecular architecture of the N‐type ATPase rotor ring from Burkholderia pseudomallei

The genome of the highly infectious bacterium Burkholderia pseudomallei harbors an atp operon that encodes an N‐type rotary ATPase, in addition to an operon for a regular F‐type rotary ATPase. The molecular architecture of N‐type ATPases is unknown and their biochemical properties and cellular functions are largely unexplored. We studied the B. pseudomallei N₁N_o‐type ATPase and investigated the structure and ion specificity of its membrane‐embedded c‐ring rotor by single‐particle electron cryo‐microscopy. Of several amphiphilic compounds tested for solubilizing the complex, the choice of the low‐density, low‐CMC detergent LDAO was optimal in terms of map quality and resolution. The cryoEM map of the c‐ring at 6.1 Å resolution reveals a heptadecameric oligomer with a molecular mass of ~141 kDa. Biochemical measurements indicate that the c₁₇ ring is H⁺ specific, demonstrating that the ATPase is proton‐coupled. The c₁₇ ring stoichiometry results in a very high ion‐to‐ATP ratio of 5.7. We propose that this N‐ATPase is a highly efficient proton pump that helps these melioidosis‐causing bacteria to survive in the hostile, acidic environment of phagosomes.     

The Mechanism of Cu+ Transport ATPases: INTERACTION WITH CU+ CHAPERONES AND THE ROLE OF TRANSIENT METAL-BINDING SITES*

Cu⁺-ATPases are membrane proteins that couple the hydrolysis of ATP to the efflux of cytoplasmic Cu⁺. In cells, soluble chaperone proteins bind and distribute cytoplasmic Cu⁺, delivering the ion to the transmembrane metal-binding sites in the ATPase. The structure of Legionella pneumophila Cu⁺-ATPase (Gourdon, P., Liu, X. Y., Skjørringe, T., Morth, J. P., Møller, L. B., Pedersen, B. P., and Nissen, P. (2011) Nature 475, 59–64) shows that a kinked transmembrane segment forms a “platform” exposed to the cytoplasm. In addition, neighboring invariant Met, Asp, and Glu are located at the “entrance” of the ion path. Mutations of amino acids in these regions of the Archaeoglobus fulgidus Cu⁺-ATPase CopA do not affect ATPase activity in the presence of Cu⁺ free in solution. However, Cu⁺ bound to the corresponding chaperone (CopZ) could not activate the mutated ATPases, and in parallel experiments, CopZ was unable to transfer Cu⁺ to CopA. Furthermore, mutation of a specific electronegative patch on the CopZ surface abolishes the ATPase activation and Cu⁺ transference, indicating that the region is required for the CopZ-CopA interaction. Moreover, the data suggest that the interaction is driven by the complementation of the electropositive platform in the ATPase and the electronegative Cu⁺ chaperone. This docking likely places the Cu⁺ proximal to the conserved carboxyl and thiol groups in the entrance site that induce metal release from the chaperone via ligand exchange. The initial interaction of Cu⁺ with the pump is transient because Cu⁺ is transferred from the entrance site to transmembrane metal-binding sites involved in transmembrane translocation.     

Coproporphyrin III excretion identifies the anaerobic coproporphyrinogen III oxidase HemN as a copper target in the Cu+‐ATPase mutant copA− of Rubrivivax gelatinosus

Two genes encoding structurally similar Copper P_1B‐type ATPases can be identified in several genomes. Notwithstanding the high sequence and structural similarities these ATPases held, it has been suggested that they fulfil distinct physiological roles. In deed, we have shown that the Cu⁺‐ATPase CtpA is required only for the activity of cuproproteins in the purple bacterium Rubrivivax gelatinosus; herein, we show that CopA is not directly required for cytochrome c oxidase but is vital for copper tolerance. Interestingly, excess copper in the copA^? mutant resulted in a substantial decrease of the cytochrome c oxidase and the photosystem under microaerobic and anaerobic conditions together with the extrusion of coproporphyrin III. The data indicated that copper targeted the tetrapyrrole biosynthesis pathway at the level of the coproporphyrinogen III oxidase HemN and thereby affects the oxidase and the photosystem. This is the first in vivo demonstration that copper, like oxygen, affects tetrapyrrole biosynthesis presumably at the level of the SAM and [4Fe‐4S] containing HemN enzyme. In light of these results and similar findings in Escherichia coli, the potential role of copper ions in the evolution of [4Fe‐4S] enzymes and the Cu⁺‐ATPases is discussed.     

Functional characterization of Legionella pneumophila Cu+ transport ATPase. The activation by Cu+ and ATP

Cu⁺-ATPases are integral membrane proteins belonging to the IB subfamily of the P-type ATPases that couple Cu⁺ transport to the hydrolysis of ATP. As some structural and functional particularities arise for Cu⁺-ATPases, several authors suggest that some of the reaction steps of the Albers-Post model postulated for other P-ATPases may be different. In this work we describe a functional characterization of Legionella pneumophila Cu⁺-ATPase (LpCopA), the first P_IB-ATPase whose structure was determined by X-ray crystallography. Cu⁺-ATPase activity of the enzyme presents a maximum at ～37 °C and pH 6.6–6.8. Phospholipids enhance LpCopA Cu⁺-ATPase activity in a non-essential mode where optimal activity is achieved at an asolectin molar fraction of 0.15 and an amphiphile-protein ratio of ~30,000. As described for other P-ATPases, Mg²⁺ acts as an essential activator. Furthermore, Cu⁺-ATPase activity dependence on [Cu⁺] and [ATP] can both be described by a sum of two hyperbolic functions. Based on that, and the [Cu⁺] and [ATP] dependencies of the best fitting parameters of the hyperbolae pointed above, we propose a minimal reaction scheme for the catalytic mechanism that shares the basic reaction steps of the Albers-Post model for P-type ATPases. The reaction scheme postulated contemplates two different binding affinities for a single ATP (apparent affinities of 0.66 and 550 μM at [Cu⁺] → ∞) and binding of at least 2 Cu⁺ with different affinities as well (apparent affinities of 1.4 and 102.5 μM at [ATP] → ∞).     

Mechanism of ATPase-mediated Cu+ Export and Delivery to Periplasmic Chaperones: THE INTERACTION OF ESCHERICHIA COLI CopA AND CusF*

Cellular copper homeostasis requires transmembrane transport and compartmental trafficking while maintaining the cell essentially free of uncomplexed Cu^2+/+. In bacteria, soluble cytoplasmic and periplasmic chaperones bind and deliver Cu⁺ to target transporters or metalloenzymes. Transmembrane Cu⁺-ATPases couple the hydrolysis of ATP to the efflux of cytoplasmic Cu⁺. Cytosolic Cu⁺ chaperones (CopZ) interact with a structural platform in Cu⁺-ATPases (CopA) and deliver copper into the ion permeation path. CusF is a periplasmic Cu⁺ chaperone that supplies Cu⁺ to the CusCBA system for efflux to the extracellular milieu. In this report, using Escherichia coli CopA and CusF, direct Cu⁺ transfer from the ATPase to the periplasmic chaperone was observed. This required the specific interaction of the Cu⁺-bound form of CopA with apo-CusF for subsequent metal transfer upon ATP hydrolysis. As expected, the reverse Cu⁺ transfer from CusF to CopA was not observed. Mutation of CopA extracellular loops or the electropositive surface of CusF led to a decrease in Cu⁺ transfer efficiency. On the other hand, mutation of Met and Glu residues proposed to be part of the metal exit site in the ATPase yielded enzymes with lower turnover rates, although Cu⁺ transfer was minimally affected. These results show how soluble chaperones obtain Cu⁺ from transmembrane transporters. Furthermore, by explaining the movement of Cu⁺ from the cytoplasmic pool to the extracellular milieu, these data support a mechanism by which cytoplasmic Cu⁺ can be precisely directed to periplasmic targets via specific transporter-chaperone interactions.     

Structural Insights into the Nucleotide-Binding Domains of the P1B-type ATPases HMA6 and HMA8 from Arabidopsis thaliana

Copper is a crucial ion in cells, but needs to be closely controlled due to its toxic potential and ability to catalyse the formation of radicals. In chloroplasts, an important step for the proper functioning of the photosynthetic electron transfer chain is the delivery of copper to plastocyanin in the thylakoid lumen. The main route for copper transport to the thylakoid lumen is driven by two P_IB-type ATPases, Heavy Metal ATPase 6 (HMA6) and HMA8, located in the inner membrane of the chloroplast envelope and in the thylakoid membrane, respectively. Here, the crystal structures of the nucleotide binding domain of HMA6 and HMA8 from Arabidopsis thaliana are reported at 1.5Å and 1.75Å resolution, respectively, providing the first structural information on plants Cu⁺-ATPases. The structures reveal a compact domain, with two short helices on both sides of a twisted beta-sheet. A double mutant, aiding in the crystallization, provides a new crystal contact, but also avoids an internal clash highlighting the benefits of construct modifications. Finally, the histidine in the HP motif of the isolated domains, unable to bind ATP, shows a side chain conformation distinct from nucleotide bound structures.     

Interactions of PAN's C‐termini with archaeal 20S proteasome and implications for the eukaryotic proteasome–ATPase interactions

Protein degradation in the 20S proteasome is regulated in eukaryotes by the 19S ATPase complex and in archaea by the homologous PAN ATPase ring complex. Subunits of these hexameric ATPases contain on their C‐termini a conserved hydrophobic‐tyrosine‐X (HbYX) motif that docks into pockets in the 20S to stimulate the opening of a gated substrate entry channel. Here, we report the crystal structure of the archaeal 20S proteasome in complex with the C‐terminus of the archaeal proteasome regulatory ATPase, PAN. This structure defines the detailed interactions between the critical C‐terminal HbYX motif and the 20S α‐subunits and indicates that the intersubunit pocket in the 20S undergoes an induced‐fit conformational change on binding of the HbYX motif. This structure together with related mutagenesis data suggest how in eukaryotes certain proteasomal ATPases bind to specific pockets in an asymmetrical manner to regulate gate opening.     

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.     

DKWSLLL,a Versatile DXXXLL‐Type Signal with Distinct Roles in the Cu+‐Regulated Trafficking of ATP7B

In the liver, the P‐type ATPase and membrane pump ATP7B plays a crucial role in Cu⁺ donation to cuproenzymes and in the elimination of excess Cu⁺. ATP7B is endowed with a COOH‐cytoplasmic (DE)XXXLL‐type traffic signal. We find that accessory (Lys ?3, Trp ?2, Ser ?1 and Leu +2) and canonical (D ?4, Leu 0 and Leu +1) residues confer the DKWSLLL signal with the versatility required for the Cu⁺‐regulated cycling of ATP7B between the trans‐Golgi network (TGN) and the plasma membrane (PM). The separate mutation of these residues caused a disruption of the signal, resulting in different ATP7B distribution phenotypes. These phenotypes indicate the key roles of specific residues at separate steps of ATP7B trafficking, including sorting at the TGN, transport from the TGN to the PM and its endocytosis, and recycling to the TGN and PM. The distinct roles of ATP7B in the TGN and PM and the variety of phenotypes caused by the mutation of the canonical and accessory residues of the DKWSLLL signal can explain the separate or joined presentation of Wilson's cuprotoxicosis and the dysfunction of the cuproenzymes that accept Cu⁺ at the TGN.      

Expression and mutagenesis of ZntA, a zinc-transporting P-type ATPase from Escherichia coli.

Cation-transporting P-type ATPases comprise a major membrane protein family, the members of which are found in eukaryotes, eubacteria, and archaea. A phylogenetically old branch of the P-type ATPase family is involved in the transport of heavy-metal ions such as copper, silver, cadmium, and zinc. In humans, two homologous P-type ATPases transport copper. Mutations in the human proteins cause disorders of copper metabolism known as Wilson and Menkes diseases. E. coli possesses two genes for heavy-metal translocating P-type ATPases. We have constructed an expression system for one of them, ZntA, which encodes a 732 amino acid residue protein capable of transporting Zn(2+). A vanadate-sensitive, Zn(2+)-dependent ATPase activity is present in the membrane fraction of our expression strain. In addition to Zn(2+), the heavy-metal ions Cd(2+), Pb(2+), and Ag(+) activate the ATPase. Incubation of membranes from the expression strain with [gamma-(33)P]ATP in the presence of Zn(2+), Cd(2+), or Pb(2+) brings about phosphorylation of two membrane proteins with molecular masses of approximately 90 and 190 kDa, most likely representing the ZntA monomer and dimer, respectively. Although Cu(2+) can stimulate phosphorylation by [gamma-(33)P]ATP, it does not activate the ATPase. Cu(2+) also prevents the Zn(2+) activation of the ATPase when present in 2-fold excess over Zn(2+). Ag(+) and Cu(+) appear not to promote phosphorylation of the enzyme. To study the effects of Wilson disease mutations, we have constructed two site-directed mutants of ZntA, His475Gln and Glu470Ala, the human counterparts of which cause Wilson disease. Both mutants show a reduced metal ion stimulated ATPase activity (about 30-40% of the wild-type activity) and are phosphorylated much less efficiently by [gamma-(33)P]ATP than the wild type. In comparison to the wild type, the Glu470Ala mutant is phosphorylated more strongly by [(33)P]P(i), whereas the His475Gln mutant is phosphorylated more weakly. These results suggest that the mutation His475Gln affects the reaction with ATP and P(i) and stabilizes the enzyme in a dephosphorylated state. The Glu470Ala mutant seems to favor the E2 state. We conclude that His475 and Glu470 play important roles in the transport cycles of both the Wilson disease ATPase and ZntA.     

The c‐ring ion binding site of the ATP synthase from Bacillus pseudofirmus OF4 is adapted to alkaliphilic lifestyle

In the c‐ring rotor of ATP synthases ions are shuttled across the membrane during ATP synthesis by a unique rotary mechanism. We investigated characteristics of the c‐ring from the alkaliphile Bacillus pseudofirmus OF4 with respect to evolutionary adaptations to operate with protons at high environmental pH. The X‐ray structures of the wild‐type c₁₃ ring at pH 9.0 and a ‘neutralophile‐like’ mutant (P51A) at pH 4.4, at 2.4 and 2.8 Å resolution, respectively, reveal a dependency of the conformation and protonation state of the proton‐binding glutamate (E⁵⁴) on environmental hydrophobicity. Faster labelling kinetics with the inhibitor dicyclohexylcarbodiimide (DCCD) demonstrate a greater flexibility of E⁵⁴ in the mutant due to reduced water occupancy within the H⁺ binding site. A second ‘neutralophile‐like’ mutant (V21N) shows reduced growth at high pH, which is explained by restricted conformational freedom of the mutant's E⁵⁴ carboxylate. The study directly connects subtle structural adaptations of the c‐ring ion binding site to in vivo effects of alkaliphile cell physiology.     

Probing the activity of a recombinant Zn2+‐transporting P‐type ATPase

P‐type ATPase proteins maintain cellular homeostasis and uphold critical concentration gradients by ATP‐driven ion transport across biological membranes. Characterization of single‐cycle dynamics by time‐resolved X‐ray scattering techniques in solution could resolve structural intermediates not amendable to for example crystallization or cryo‐electron microscopy sample preparation. To pave way for such time‐resolved experiments, we used biochemical activity measurements, Attenuated Total Reflectance (ATR) and time‐dependent Fourier‐Transform Infra‐Red (FTIR) spectroscopy to identify optimal conditions for activating a Zn²⁺‐transporting Type‐I ATPase from Shigella sonnei (ssZntA) at high protein concentration using caged ATP. The highest total activity was observed at a protein concentration of 25 mg/mL, at 310 K, pH 7, and required the presence of 20% (v/v) glycerol as stabilizing agent. Neither the presence of caged ATP nor increasing lipid‐to‐protein ratio affected the hydrolysis activity significantly. This work also paves way for characterization of recombinant metal‐transporting (Type‐I) ATPase mutants with medical relevance.