Characterization of the soluble domain of the ABC7 type transporter Atm1

Atm1 is an ABC transporter that is located in yeast mitochondria and has previously been implicated in the maturation of cytosolic iron-sulfur cluster proteins. The soluble nucleotide binding domain of Atm1 (Atm1-C) has been overexpressed in Escherichia coli, purified, and characterized. Dissociation constants (KD) for Atm1-C binding of ATP (KD approximately 97 microm, pH 7.3, and approximately 102 microm, pH 10.0) and ADP (KD approximately 43 microm, pH 7.3, and 92 microm, pH 10.0) were measured by fluorimetry. The higher binding affinity for ADP suggests that the transmembrane-spanning domain may be required to promote a structural change in the nucleotide binding domain to facilitate substrate export and ADP release. ADP also had an inhibitory effect on Atm1-C with an IC50 of 10 mm. The Michaelis-Menten constants Vmax, KM, and kcat of Atm1-C were measured as 1.822 microm min(-1), 513 microm, and 0.055 min(-1), respectively. The metal dependence of Atm1-C ATPase demonstrated a reactivity order of Mn2+ > Mg2+ > Co2+, while Mg2+ and Co2+ were both found to be inhibitory at higher concentrations. The pH profile and structural comparison with HisP are consistent with a role for His and Lys in promoting the ATPase activity. Structural analysis of Atm1-C by CD spectroscopy suggested a similarity of secondary structure to that found for a prokaryotic homologue (HisP), whereas modeling of the Atm1-C tertiary structure using HisP as a template is also consistent with a similarity in tertiary structure. Atm1-C tends to form a dimer or higher aggregation state at higher concentration; however, the concentration dependence of Atm1-C on ATPase activity and the results of a Hill analysis (napp = 1.1) demonstrated that there was essentially no cooperativity in ATP hydrolysis, in contrast to observations for the prokaryotic HisP transporter, which demonstrated full cooperativity for both full-length and the soluble domains. Accordingly, any cooperative response must be mediated through the transmembrane domain in the case of the eukaryotic Atm1 transporter.     

Nucleotide-dependent conformational changes in HisP: molecular dynamics simulations of an ABC transporter nucleotide-binding domain

ATP-binding cassette (ABC) transporters mediate the movement of molecules across cell membranes in both prokaryotes and eukaryotes. In ABC transporters, solute translocation occurs after ATP is either bound or hydrolyzed at the intracellular nucleotide-binding domains (NBDs). Molecular dynamics (MD) simulations have been employed to study the interactions of nucleotide with NBD. The results of extended (approximately 20 ns) MD simulations of HisP (total simulation time approximately 80 ns), the NBD of the histidine transporter HisQMP2J from Salmonella typhimurium, are presented. Analysis of the MD trajectories reveals conformational changes within HisP that are dependent on the presence of ATP in the binding pocket of the protein, and are sensitive to the presence/absence of Mg ions bound to the ATP. These changes are predominantly confined to the alpha-helical subdomain of HisP. Specifically there is a rotation of three alpha-helices within the subdomain, and a movement of the signature sequence toward the bound nucleotide. In addition, there is considerable conformational flexibility in a conserved glutamine-containing loop, which is situated at the interface between the alpha-helical subdomain and the F1-like subdomain. These results support the mechanism for ATP-induced conformational transitions derived from the crystal structures of other NBDs.     

Nonequivalence of the nucleotide-binding subunits of an ABC transporter, the histidine permease, and conformational changes in the membrane complex

The membrane-bound complex of the Salmonella typhimurium histidine permease, an ABC transporter (or traffic ATPase), is composed of two membrane proteins, HisQ and HisM, and two identical copies of an ATP-hydrolyzing protein, HisP. We have developed a technique that monitors quantitatively the sulfhydryl modification levels within the intact complex, and we have used it to investigate whether the HisP subunits behave identically within the complex. We show here that they interact differently with various thiol-specific reagents, thus indicating that, despite being identical, they are arranged asymmetrically. The possible basis of this asymmetry is discussed. We have also analyzed the occurrence of conformational changes during various stages of the activity cycle using thiol-specific reagents, fluorescence measurements, and circular dichroism spectroscopy. Cys-51, located close to the ATP-binding pocket, reflects conformational changes upon binding of ATP but does not participate in changes involved in signaling and translocation. The latter are shown to cause secondary structure alterations, as indicated by changes in alpha-helices; tertiary structure alterations also occur, as shown by fluorescence studies.     

Modulation of ATPase activity by physical disengagement of the ATP-binding domains of an ABC transporter, the histidine permease.

The membrane-bound complex of the prokaryotic histidine permease, a periplasmic protein-dependent ABC transporter, is composed of two hydrophobic subunits, HisQ and HisM, and two identical ATP-binding subunits, HisP, and is energized by ATP hydrolysis. The soluble periplasmic binding protein, HisJ, creates a signal that induces ATP hydrolysis by HisP. The crystal structure of HisP has been resolved and shown to have an "L" shape, with one of its arms (arm I) being involved in ATP binding and the other one (arm II) being proposed to interact with the hydrophobic subunits (Hung, L.-W., Wang, I. X., Nikaido, K., Liu, P.-Q., Ames, G. F.-L., and Kim, S.-H. (1998) Nature 396, 703-707). Here we study the basis for the defect of several HisP mutants that have an altered signaling pathway and hydrolyze ATP constitutively. We use biochemical approaches to show that they produce a loosely assembled membrane complex, in which the mutant HisP subunits are disengaged from HisQ and HisM, suggesting that the residues involved are important in the interaction between HisP and the hydrophobic subunits. In addition, the mutant HisPs are shown to have lower affinity for ADP and to display no cooperativity for ATP. All of the residues affected in these HisP mutants are located in arm II of the crystal structure of HisP, thus supporting the proposed function of arm II of HisP as interacting with HisQ and HisM. A revised model involving a cycle of disengagement and reengagement of HisP is proposed as a general mechanism of action for ABC transporters.     

One intact ATP-binding subunit is sufficient to support ATP hydrolysis and translocation in an ABC transporter, the histidine permease.

The membrane-bound complex of the Salmonella typhimurium histidine permease, a member of the ABC transporters (or traffic ATPases) superfamily, is composed of two integral membrane proteins, HisQ and HisM, and two copies of an ATP-binding subunit, HisP, which hydrolyze ATP, thus supplying the energy for translocation. The three-dimensional structure of HisP has been resolved. Extensive evidence indicates that the HisP subunits form a dimer. We investigated the mechanism of action of such a dimer, both within the complex and in soluble form, by creating heterodimers between the wild type and mutant HisP proteins. The data strongly suggest that within the complex both subunits hydrolyze ATP and that one subunit is activated by the other. In a heterodimer containing one wild type and one hydrolysis defective subunit both hydrolysis and ligand translocation occur at half the rate of the wild type. Soluble HisP also hydrolyzes ATP if one subunit is inactive; its specific activity is identical to that of the wild type, indicating that only one of the subunits in a soluble dimer is involved in hydrolysis. We show that the activating ability varies depending on the nature of the substitution of a well conserved residue, His-211.     

Characterization of ATPase activity of recombinant human Pif1

Saccharomyces cerevisiae Piflp helicase is the founding member of the Pifl subfamily that isconserved from yeast to human.The potential human homolog of the yeast PIFI gene has been cloned fromthe cDNA library of the Hek293 cell line.Here,we described a purification procedure of glutathione S-transferase(GST)-fused N terminal truncated human Pifl protein(hPif1ΔN)from yeast and characterizedthe enzymatic kinetics of its ATP hydrolysis activity.The ATPase activity of human Pif1 is dependent ondivalent cation,such as Mg~(2 ),Ca~(2 )and single-stranded DNA.Km for ATP for the ATPase activity isapproximately 200 μM.As the ATPase activity is essential for hPifl's helicase activity,these results willfacilitate the further investigation on hPif1.     

Purification and characterization of a deoxyribonucleic acid dependent adenosinetriphosphatase from mouse FM3A cells: effects of ribonucleoside triphosphates on the interaction of the enzyme with single-stranded DNA

There are at least four forms of DNA-dependent ATPase in mouse FM3A cells [Tawaragi, Y., Enomoto, T., Watanabe, Y., Hanaoka, F., & Yamada, M. (1984) Biochemistry 23, 529-533]. One of these, ATPase B, has been purified and characterized in detail. During the purification of the enzyme, we encountered the difficulties that the enzyme could not be recovered well from the single-stranded DNA-cellulose column and that the enzyme activity was distributed very broadly. The problems were resolved by the addition of ATP in the elution buffer. The ATPase has a sedimentation coefficient of 5.5 S in both high salt and low salt. The enzyme hydrolyzes rNTPs and dATP, but ATP and dATP are preferred substrates. Adenosine 5'-O-(3-thiotriphosphate) (ATP-gamma-S), 5'-adenylyl methylenediphosphate (AMP-PCP), and 5'-adenylyl imidodiphosphate (AMP-PNP) inhibit the enzyme activity. The enzyme is insensitive to ouabain, oligomycin, novobiocin, and ethidium bromide. A divalent cation (Mg2+ congruent to Mn2+ greater than Ca2+) as well as a nucleic acid cofactor is required for activity. Poly(dT), single-stranded circular DNA, and heat-denatured DNA were very effective. Native DNA was little effective with an efficiency of 29% of that obtained with heat-denatured DNA. In addition, the enzyme showed almost no activity with poly(dA).poly(dT) although it showed very high activity with the noncomplementary combination of poly(dT) and poly(dC), suggesting that ATPase B requires single-stranded DNA for activity. ATP altered the affinity of ATPase B for single-stranded DNA. The interaction of the enzyme with DNA was studied by Sephadex G-200 gel filtration assay.(ABSTRACT TRUNCATED AT 250 WORDS)     

Contribution of Msh2 and Msh6 subunits to the asymmetric ATPase and DNA mismatch binding activities of Saccharomyces cerevisiae Msh2-Msh6 mismatch repair protein

Previous analyses of both Thermus aquaticus MutS homodimer and Saccharomyces cerevisiae Msh2-Msh6 heterodimer have revealed that the subunits in these protein complexes bind and hydrolyze ATP asymmetrically, emulating their asymmetric DNA binding properties. In the MutS homodimer, one subunit (S1) binds ATP with high affinity and hydrolyzes it rapidly, while the other subunit (S2) binds ATP with lower affinity and hydrolyzes it at an apparently slower rate. Interaction of MutS with mismatched DNA results in suppression of ATP hydrolysis at S1-but which of these subunits, S1 or S2, makes specific contact with the mismatch (e.g., base stacking by a conserved phenylalanine residue) remains unknown. In order to answer this question and to clarify the links between the DNA binding and ATPase activities of each subunit in the dimer, we made mutations in the ATPase sites of Msh2 and Msh6 and assessed their impact on the activity of the Msh2-Msh6 heterodimer (in Msh2-Msh6, only Msh6 makes base specific contact with the mismatch). The key findings are: (a) Msh6 hydrolyzes ATP rapidly, and thus resembles the S1 subunit of the MutS homodimer, (b) Msh2 hydrolyzes ATP at a slower rate, and thus resembles the S2 subunit of MutS, (c) though itself an apparently weak ATPase, Msh2 has a strong influence on the ATPase activity of Msh6, (d) Msh6 binding to mismatched DNA results in suppression of rapid ATP hydrolysis, revealing a "cis" linkage between its mismatch recognition and ATPase activities, (e) the resultant Msh2-Msh6 complex, with both subunits in the ATP-bound state, exhibits altered interactions with the mismatch.     

The nucleotide-binding site of HisP, a membrane protein of the histidine permease. Identification of amino acid residues photoaffinity labeled by 8-azido-ATP

The periplasmic histidine transport system (permease) of Escherichia coli and Salmonella typhimurium is composed of a soluble, histidine-binding receptor located in the periplasm and a complex of three membrane-bound proteins of which one, HisP, was shown previously to bind ATP. These permeases are energized by ATP. HisP is a member of a family of membrane transport proteins which is conserved in all periplasmic permeases and is presumed to be involved in coupling the energy of ATP to periplasmic transport. In this paper the nature of the ATP-binding site of HisP has been explored by identification of some of the residues that come into contact with ATP. HisP was derivatized with 8-azido-ATP (N3ATP). Both the underivatized and the derivatized forms of HisP were solubilized, purified, and digested with trypsin. The resulting tryptic peptides were resolved by high pressure liquid chromatography, and peptides modified by N3ATP were isolated and sequenced. Two peptides, X and Z, spanning amino acid residues 16-23 and 31-45, were found to contain sites of N3ATP attachment at His19 and Ser41, respectively. Both peptides are close to the amino-terminal end of HisP; peptide Z is located in one of the well conserved regions comprising the nucleotide-binding consensus motifs of the energy-coupling components of these permeases. These consensus motifs are found in many purine nucleotide-binding proteins. The relationship between the location of these residues and the overall structure of the ATP-binding site is discussed.     

Ultrafast purification and reconstitution of His-tagged cysteine-less Escherichia coli F1Fo ATP synthase

His-tagged cysteine-less F1Fo ATP synthase from Escherichia coli was purified using Ni-NTA affinity chromatography. During the purification procedure the loss of total ATPase activity did not exceed 50%, and the extent of purification was about 80-fold. The purified enzyme was essentially free of other proteins, was highly active in ATP hydrolysis (75 units/mg at pH 8 and 37 degrees C), and was sensitive to N,N'-dicyclohexylcarbodiimide (70%). Incorporation of F1Fo into soybean liposomes yielded well-coupled and highly active proteoliposomes. The entire procedure, from the disruption of cells by French press to the preparation of proteoliposomes, took only about 8 h. Some improvements in procedures for the estimation of rates of both ATP hydrolysis and ATP-dependent 9-amino-6-chloro-2-methoxyacridine (ACMA) fluorescence quenching are described.     

A DNA dependent ATPase from HeLa cells

The purification and the properties of the major DNA dependent ATPase from HeLa cells are described. This enzyme is present in the nucleus and in the cytoplasm in approximately equal amounts. It has a Mr of about 110000 dalton and it hydrolyzes ATP (and dATP) to ADP+Pi only in the presence of single-stranded DNA. The enzyme shows an ATP dependent unwinding activity on DNA duplex, with a 3′ to 5′ polarity of the unwound strand. Under certain conditions the enzyme is able to stimulate the activity of DNA polymeraseα on appropriate DNA templates. Such stimulation is synergistic with that exerted by a DNA binding protein from calf thymus.     

Dominant Saccharomyces cerevisiae msh6 mutations cause increased mispair binding and decreased dissociation from mispairs by Msh2-Msh6 in the presence of ATP

A previous study described four dominant msh6 mutations that interfere with both the Msh2-Msh6 and Msh2-Msh3 mismatch recognition complexes (Das Gupta, R., and Kolodner, R. D. (2000) Nat. Genet. 24, 53-56). Modeling predicted that two of the amino acid substitutions (G1067D and G1142D) interfere with protein-protein interactions at the ATP-binding site-associated dimer interface, one (S1036P) similarly interferes with protein-protein interactions and affects the Msh2 ATP-binding site, and one (H1096A) affects the Msh6 ATP-binding site. The ATPase activity of the Msh2-Msh6-G1067D and Msh2-Msh6-G1142D complexes was inhibited by GT, +A, and +AT mispairs, and these complexes showed increased binding to GT and +A mispairs in the presence of ATP. The ATPase activity of the Msh2-Msh6-S1036P complex was inhibited by a GT mispair, and it bound the GT mispair in the presence of ATP, whereas its interaction with insertion mispairs was unchanged compared with the wild-type complex. The ATPase activity of the Msh2-Msh6-H1096A complex was generally attenuated, and its mispair-binding behavior was unaffected. These results are in contrast to those obtained with the wild-type Msh2-Msh6 complex, which showed mispair-stimulated ATPase activity and ATP inhibition of mispair binding. These results indicate that the dominant msh6 mutations cause more stable binding to mispairs and suggest that there may be differences in how base base and insertion mispairs are recognized.     

Studies on the structure and conformation of yeast mitochondrial ATPase using aurovertin and methanol as probes

1. The isolation of the mitochondrial ATPase F1 and its beta-subunit from commercial baker's yeast (Saccharomyces cerevisiae) is described. 2. The molecular weight determined by ultracentrifugation is 340000 +/- 30000. Gel chromatography indicates a molecular weight of 300000 +/- 20000. 3. Fluorimetric titration of the isolated enzyme with aurovertin reveals two binding sites per molecule. The isolated beta-subunit binds aurovertin in a 1 : 1 stoicheiometry. It is concluded that the ATPase molecule contains two aurovertin-binding beta-subunits. 4. The stabilizing agent methanol influences both the measured Kd and the concentration of binding sites for aurovertin. These results fit a model in which both F1 and aurovertin are distributed between aqueous and methanol phases. 5. The effect of methanol on the ATPase activity can be described in terms of the model proposed by Recktenwald and Hess (Recktenwald, D. and Hess, B. (1977) FEBS Lett. 76, 25-28). It is proposed that methanol enhances the affinity of the regulatory site for ATP, but at higher concentrations prevents the interaction between the regulatory and catalytic sites. 6. Since HSO(-3), a typical effector of the assumed regulatory site of F1, has no effect on the binding of aurovertin, it is concluded that the binding site of aurovertin is not correlated with the regulatory site. 7. The inhibition of ATPase activity by aurovertin is slowly (t 1/2 = 70 s) induced during turnover conditions. 8. From the effect of methanol on the inhibition of ATPase activity by aurovertin it is concluded that under turnover conditions the conformation is such that the aurovertin-binding sites have a 6-fold higher affinity for methanol than under resting conditions.     

Evidence for the occurrence of an ecto-(adenosine triphosphatase) in rat epididymal spermatozoa.

Intact spermatozoa from rat cauda epididymis possess a Mg2+-dependent ATPase activity that hydrolyses externally added [gamma-32P]ATP. The ATPase reaction was linear with time for approx. 6 min and there was no detectable uptake of ATP by these cells. The ATPase activity of the whole spermatozoa was not due to leakage of the intracellular enzymic activity, contamination of the broken cells or any possible cell damage during incubation and isolation of spermatozoa. The activity of the enzyme was strongly inhibited (approx. 85%) by p-chloromercuribenzenesulphonic acid (50 microM) or the diazonium salt of sulphanilic acid (50 microM), which are believed not to enter the cells, whereas ouabain (0.5 mM), NaF (10 mM), NaN3 (2.5 mM) and oligomycin (5 microM) had no appreciable effect on the activity of the spermatozoal APTase. There was little loss of ATPase activity from the cells when washed with 0.5 mM-EDTA and an iso-osmotic or hyperosmotic medium. These data are consistent with the view that the observed ATPase activity is located on the external surface of spermatozoa. The sperm ecto-ATPase activity is resistant to the action of proteinases (50 micrograms/ml), namely trypsin, chymotrypsin and Pronase. Studies with various unlabelled phosphate esters indicate that the sperm ecto-ATPase is not a non-specific phosphatase and it has high degree of substrate specificity for ATP.     

Functional consequences of mutations in the conserved 'signature sequence' of the ATP-binding-cassette protein MalK.

The binding-protein-dependent maltose-transport system of enterobacteria, a member of the ATP-binding-cassette (ABC) transporter superfamily, is composed of two integral membrane proteins, MalF and MalG, and two copies of an ATPase subunit, MalK, which hydrolyze ATP, thus energizing the translocation process. Isolated MalK displays spontaneous ATPase activity, whereas in the assembled MalFGK2 complex, reconstituted in liposomes, ATP hydrolysis requires stimulation by the substrate-loaded extracellular maltose-binding protein, MalE. The ATPase domains of ABC transporters, including MalK, share a unique sequence motif ('LSGGQ', 'signature sequence' or 'linker peptide') with as yet unknown function. To elucidate its role in the transport process, we investigated the consequences of mutations affecting two highly conserved residues (G137, Q140) in the MalK-ATPase of Salmonella typhimurium, by biochemical means. Residues corresponding to Q140 in other ABC proteins have not yet been studied. All mutant alleles (G137--> A, V, T; Q140--> L, K, N) fail to restore a functional transport complex in vivo. In addition, the mutations increase the repressing activity of MalK on other maltose-regulated genes when compared with wild-type MalK. Purified variants of G137 have lost the ability to hydrolyze ATP but still display nucleotide-binding activity, albeit with reduced affinity. Binding of MgATP results in similar protection against trypsin, as observed with wild-type, indicating no major change in protein structure. In contrast, the variants of Q140 differ in their properties, depending on the chemical nature of the replacement residue. MalKQ140L fails to hydrolyze ATP and exhibits a strong intrinsic resistance to trypsin in the absence of MgATP, suggesting a drastically altered conformation. In contrast, the purified mutant proteins Q140K and Q140N display ATPase activities and MgATP-induced changes in the tryptic cleavage pattern similar to those of wild-type. However, mutant transport complexes containing the Q140K or Q140N variants, when studied in proteoliposomes, are severely impaired in MalE-maltose-stimulated ATPase activity. These results are discussed with respect to the crystal structure of the homologous HisP protein [Hung, L.-W., Wang, I.X., Nikaido, K., Liu, P.-Q., Ames, G.F.-L. & Kim, S.-H. (1998) Nature (London) 396, 703-707] and are interpreted in favor of a role of the signature sequence in activating the hydrolyzing activity of MalK upon substrate-initiated conformational changes in MalF/MalG.     

Mitochondrial adenosine triphosphatase from human placenta--purification and catalytic properties

1. The purification of ATPase (EC 3.6.1.3) from human placental mitochondria is described. The yield based on mitochondrial enzyme activity was about 70% and the purification was 380-fold. 2. The rate of Mg-ATP hydrolysis was 85 mumole per min per mg of protein under optimum conditions. 3. Nucleoside triphosphates were hydrolyzed by the purified enzyme at decreasing rates in the following order: GTP greater than ITP greater than ATP greater than epsilon-ATP greater than UTP greater than CTP in Tris-HCl buffer (pH 8.0), and in the order: ATP greater than GTP greater than or equal to ITP greater than epsilon-ATP greater than UTP greater than CTP in Tris-bicarbonate buffer at pH 8.0. 4. The values of kinetic parameters are reported. The ATPase reaction deviated from typical Michaelis-Menten kinetics in Tris-HCl buffer but not in Tris-bicarbonate. Eadie-Hofstee plots for Mg-ATP hydrolysis were biphasic in Tris-HCl (Km = 0.2 mM, 0.09 mM) and monophastic in Tris-becarbonate medium (Km = 0.16 mM). 5. In the presence of Mg-ITP or Mg-GTP as substrates no curvature of the reciprocal plots was observed. 6. The results presented reflect the fact that multiple conformations of the enzyme molecule do exist and are probably involved in its regulatory functions. 7. The existence of two kinetically distinct classes of catalytic sites and of an anion-binding site on the placental ATPase is proposed.     

The yeast a-factor transporter Ste6p, a member of the ABC superfamily, couples ATP hydrolysis to pheromone export

ATP-binding cassette (ABC) proteins transport a diverse collection of substrates. It is presumed that these proteins couple ATP hydrolysis to substrate transport, yet ATPase activity has been demonstrated for only a few. To provide direct evidence for such activity in Ste6p, the yeast ABC protein required for the export of a-factor mating pheromone, we established conditions for purification of Ste6p in biochemical quantities from both yeast and Sf9 insect cells. The basal ATPase activity of purified and reconstituted Ste6p (V(max) = 18 nmol/mg/min; K(m) for MgATP = 0.2 mm) compares favorably with several other ABC proteins and was inhibited by orthovanadate in a profile diagnostic of ABC transporters (apparent K(I) = 12 microm). Modest stimulation (approximately 40%) was observed upon the addition of a-factor either synthetic or in native form. We also used an 8-azido-[alpha-(32)P]ATP binding and vanadate-trapping assay to examine the behavior of wild-type Ste6p and two different double mutants (G392V/G1087V and G509D/G1193D) shown previously to be mating-deficient in vivo. Both mutants displayed a diminished ability to hydrolyze ATP, with the latter uncoupled from pheromone transport. We conclude that Ste6p catalyzes ATP hydrolysis coupled to a-factor transport, which in turn promotes mating.     

Proton ATPase of rat liver mitochondria: A rapid procedure for purification of a stable, reconstitutively active F1 preparation using a modified chloroform method

A method is described for the purification of rat liver F1-ATPase by a modification of the chloroform extraction procedure originally described by Beechey et al. (Biochem. J. (1975) 148, 533). Purified liver membrane vesicles are extracted with chloroform in the presence of ATP and EDTA. The procedure yields pure F1 in only 2-3 h without the necessity of ion-exchange chromatography. The enzyme exhibits the alpha, beta, gamma, delta, and epsilon bands characteristic of F1-ATPase. It has a high ATPase specific activity, and is reconstitutively active, catalyzing high rates of ATP synthesis. Significantly, it can be readily crystallized. If desired, the enzyme can be passed over a gel filtration column to place it in a stabilizing phosphate-EDTA buffer, lyophilized and stored indefinitely at -20 degrees C.     

The metal-binding sites of the zinc-transporting P-type ATPase of Escherichia coli. Lys693 and Asp714 in the seventh and eighth transmembrane segments of ZntA contribute to the coupling of metal binding and ATPase activity

ZntA is a P-type ATPase which transports Zn(2+), Pb(2+) and Cd(2+) out of the cell. Two cysteine-containing motifs, CAAC near the N-terminus and CPC in transmembrane helix 6, are involved in binding of the translocated metal. We have studied these motifs by mutating the cysteines to serines. The roles of two other possible metal-binding residues, K(693) and D(714), in transmembrane helices 7 and 8, were also addressed. The mutation CAAC-->SAAS reduces the ATPase activity by 50%. The SAAS mutant is phosphorylated with ATP almost as efficiently as the wild type. However, its phosphorylation with P(i) is poorer than that of the wild type and its dephosphorylation rate is faster than that of the wild type ATPase. The CPC-->SPS mutant is inactive but residual phosphorylation with ATP could still be observed. The most important findings of this work deal with the prospective metal-binding residues K(693) and D(714): the substitution K693N eliminates the Zn(2+)-stimulated ATPase activity completely, although significant Zn(2+)-dependent phosphorylation by ATP remains. The K693N ATPase is hyperphosphorylated by P(i). ZntA carrying the change D714M has strong metal-independent ATPase activity and is very weakly phosphorylated both by ATP and P(i). In conclusion, K(693) and D(714) are functionally essential and appear to contribute to the metal specificity of ZntA, most probably by being parts of the metal-binding site made up by the CPC motif.     

Characterization of the adenosinetriphosphatase and transport activities of purified cystic fibrosis transmembrane conductance regulator

The cystic fibrosis transmembrane conductance regulator (CFTR) functions in vivo as a cAMP-activated chloride channel. A member of the ATP-binding cassette superfamily of membrane transporters, CFTR contains two transmembrane domains (TMDs), two nucleotide-binding domains (NBDs), and a regulatory (R) domain. It is presumed that CFTR couples ATP hydrolysis to channel gating, and as a first step in addressing this issue directly, we have established conditions for purification of biochemical quantities of human CFTR expressed in Sf9 insect cells. Use of an 8-azido[alpha-(32)P]ATP-binding and vanadate-trapping assay allowed us to devise conditions to preserve CFTR function during purification of a C-terminal His(10)-tagged variant after solubilization with lysophosphatidylglycerol (1%) and diheptanoylphosphatidylcholine (0.3%) in the presence of excess phospholipid. Study of purified and reconstituted CFTR showed that it binds nucleotide with an efficiency comparable to that of P-glycoprotein and that it hydrolyzes ATP at rates sufficient to account for presumed in vivo activity [V(max) of 58 +/- 5 nmol min(-1) (mg of protein)(-1), K(M)(MgATP) of 0.15 mM]. In further work, we found that neither nucleotide binding nor ATPase activity was altered by phosphorylation (using protein kinase A) or dephosphorylation (with protein phosphatase 2B); we also observed inhibition (approximately 40%) of ATP hydrolysis by reduced glutathione but not by DTT. To evaluate CFTR function as an anion channel, we introduced an in vitro macroscopic assay based on the equilibrium exchange of proteoliposome-entrapped radioactive tracers. This revealed a CFTR-dependent transport of (125)I that could be inhibited by known chloride channel blockers; no significant CFTR-dependent transport of [alpha-(32)P]ATP was observed. We conclude that heterologous expression of CFTR in Sf9 cells can support manufacture and purification of fully functional CFTR. This should aid in further biochemical characterization of this important molecule.