Two ATPases.

In this article, I reflect on research on two ATPases. The first is F(1)F(0)-ATPase, also known as ATP synthase. It is the terminal enzyme in oxidative phosphorylation and famous as a nanomotor. Early work on mitochondrial enzyme involved purification in large amount, followed by deduction of subunit composition and stoichiometry and determination of molecular sizes of holoenzyme and individual subunits. Later work on Escherichia coli enzyme utilized mutagenesis and optical probes to reveal the molecular mechanism of ATP hydrolysis and detailed facets of catalysis. The second ATPase is P-glycoprotein, which confers multidrug resistance, notably to anticancer drugs, in mammalian cells. Purification of the protein in large quantity allowed detailed characterization of catalysis, formulation of an alternating sites mechanism, and recently, advances in structural characterization.

Role of {alpha}-subunit VISIT-DG sequence residues Ser-347 and Gly-351 in the catalytic sites of Escherichia coli ATP synthase.

This paper describes the role of alpha-subunit VISIT-DG sequence residues alphaSer-347 and alphaGly-351 in catalytic sites of Escherichia coli F(1)F(o) ATP synthase. X-ray structures show the very highly conserved alpha-subunit VISIT-DG sequence in close proximity to the conserved phosphate-binding residues alphaArg-376, betaArg-182, betaLys-155, and betaArg-246 in the phosphate-binding subdomain. Mutations alphaS347Q and alphaG351Q caused loss of oxidative phosphorylation and reduced ATPase activity of F(1)F(o) in membranes by 100- and 150-fold, respectively, whereas alphaS347A mutation showed only a 13-fold loss of activity and also retained some oxidative phosphorylation activity. The ATPase of alphaS347Q mutant was not inhibited, and the alphaS347A mutant was slightly inhibited by MgADP-azide, MgADP-fluoroaluminate, or MgADP-fluoroscandium, in contrast to wild type and alphaG351Q mutant. Whereas 7-chloro-4-nitrobenzo-2-oxa-1, 3-diazole (NBD-Cl) inhibited wild type and alphaG351Q mutant ATPase essentially completely, ATPase in alphaS347A or alphaS347Q mutant was inhibited maximally by approximately 80-90%, although reaction still occurred at residue betaTyr-297, proximal to the alpha-subunit VISIT-DG sequence, near the phosphate-binding pocket. Inhibition characteristics supported the conclusion that NBD-Cl reacts inbetaE (empty) catalytic sites, as shown previously by x-ray structure analysis. Phosphate protected against NBD-Cl inhibition in wild type and alphaG351Q mutant but not in alphaS347Q or alphaS347A mutant. The results demonstrate that alphaSer-347 is an additional residue involved in phosphate-binding and transition state stabilization in ATP synthase catalytic sites. In contrast, alphaGly-351, although strongly conserved and clearly important for function, appears not to play a direct role.

Nucleotide-induced structural changes in P-glycoprotein observed by electron microscopy.

P-glycoprotein (Pgp) is an ATP hydrolysis driven multidrug efflux pump, which, when overexpressed in the plasma membrane of certain cancers, can lead to the failure of chemotherapy. Previously, we have presented a projection structure of nucleotide-free mouse Pgp from electron microscopic images of lipid monolayer-generated two-dimensional crystals ( Lee, J. Y., Urbatsch, I. L., Senior, A. E., and Wilkens, S. (2002) J. Biol. Chem. 277, 40125-40131 ). Here we have analyzed the structure of cysteine-free human Pgp from two-dimensional crystals that were generated with the same lipid-monolayer technique in the absence and presence of various nucleotides. The images show that human Pgp has a similar structure to the mouse protein. Furthermore, the analysis of projection structures obtained under different nucleotide conditions suggests that Pgp can exist in at least two major conformations, one of which shows a central cavity between the N- and C-terminal halves of the molecule and another in which the two halves have moved sideways, thereby closing the central cavity. Intermediate conformations were observed for some nucleotide/vanadate combinations. A low-resolution, three-dimensional model of human Pgp was calculated from tilted specimen crystallized in the presence of the non-hydrolyzable nucleotide analog, adenosine 5'-O-(thiotriphosphate). The structural analysis presented here adds to the emerging picture that multidrug ABC transporters function by switching between two major conformations in a nucleotide-dependent manner.

ATP synthase: motoring to the finish line.

Protonmotive force produced by the electron transport chain is harnessed by the rotary molecular nanomotor ATP synthase to generate ATP. In this issue of Cell, Adachi et al. (2007), in a dazzling display of technical sophistication, now disentangle the coupling between the mechanical force generated by rotation of the ATP synthase subunits and the chemical reactions that occur simultaneously at the enzyme's three catalytic sites.

Mutational analysis of conserved aromatic residues in the A-loop of the ABC transporter ABCB1A (mouse Mdr3).

The A-loop is a recently described conserved region in the NBDs of ABC transporters [Ambudkar, S.V., Kim, I.-W., Xia, D. and Sauna, Z.E. (2006) The A-loop, a novel conserved aromatic acid subdomain upstream of the Walker A motif in ABC transporters, is critical for ATP binding. FEBS Lett. 580, 1049-1055; Kim, I.W., Peng, X.H., Sauna, Z.E., FitzGerald, P.C., Xia, D., Muller, M., Nandigama, K. and Ambudkar, S.V. (2006) The conserved tyrosine residues 401 and 1044 in ATP sites of human P-glycoprotein are critical for ATP binding and hydrolysis: evidence for a conserved subdomain, the A-loop in the ATP-binding cassette. Biochemistry 45, 7605-7616]. In mouse P-glycoprotein (Abcb1a), the aromatic residue of the A-loop in both NBDs is a tyrosine: Y397 in NBD1 and Y1040 in NBD2. Another tyrosine residue (618 in NBD1 and 1263 in NBD2) also appears to lie in proximity to the ATP molecule. We have mutated residues Y397, Y618, Y1040, and Y1263 to tryptophan and analyzed the effect of these substitutions on transport properties, ATP binding, and ATP hydrolysis by Abcb1a (mouse Mdr3). Y618W and Y1263W enzymes had catalytic characteristics similar to WT Abcb1a. On the other hand, Y397W and Y1040W showed impaired transport and greatly reduced ATPase activity, including a approximately 10-fold increase in Km for MgATP. Thus, Y397 and Y1040 play an important role in Abcb1a catalysis.

Assembly of the stator in Escherichia coli ATP synthase. Complexation of alpha subunit with other F1 subunits is prerequisite for delta subunit binding to the N-terminal region of alpha.

Alpha subunit of Escherichia coli ATP synthase was expressed with a C-terminal 6-His tag and purified. Pure alpha was monomeric, was competent in nucleotide binding, and had normal N-terminal sequence. In F1 subunit dissociation/reassociation experiments it supported full reconstitution of ATPase, and reassociated complexes were able to bind to F1-depleted membranes with restoration of ATP-driven proton pumping. Therefore interaction between the stator delta subunit and the N-terminal residue 1-22 region of alpha occurred normally when pure alpha was complexed with other F1 subunits. On the other hand, three different types of experiments showed that no interaction occurred between pure delta and isolated alpha subunit. Unlike in F1, the N-terminal region of isolated alpha was not susceptible to trypsin cleavage. Therefore, during assembly of ATP synthase, complexation of alpha subunit with other F1 subunits is prerequisite for delta subunit binding to the N-terminal region of alpha. We suggest that the N-terminal 1-22 residues of alpha are sequestered in isolated alpha until released by binding of beta to alpha subunit. This prevents 1/1 delta/alpha complexes from forming and provides a satisfactory explanation of the stoichiometry of one delta per three alpha seen in the F1 sector of ATP synthase, assuming that steric hindrance prevents binding of more than one delta to the alpha3/beta3 hexagon. The cytoplasmic fragment of the b subunit (bsol) did not bind to isolated alpha. It might also be that complexation of alpha with beta subunits is prerequisite for direct binding of stator b subunit to the F1-sector.

Inhibition of the ATPase activity of Escherichia coli ATP synthase by magnesium fluoride.

Inhibition of ATPase activity of Escherichia coli ATP synthase by magnesium fluoride (MgFx) was studied. Wild-type F(1)-ATPase was inhibited potently, albeit slowly, when incubated with MgCl(2), NaF, and NaADP. The combination of all three components was required. Reactivation of ATPase activity, after removal of unbound ligands, occurred with half-time of approximately 14 h at 22 degrees C and was quasi-irreversible at 4 degrees C. Mutant F(1)-ATPases, in which catalytic site residues involved in transition state formation were modified, were found to be resistant to inhibition by MgFx. The data demonstrate that MgFx in combination with MgADP behaves as a tight-binding transition state analog in E. coli ATP synthase.

Expression, purification, and characterization of cysteine-free mouse P-glycoprotein.

Cysteine-free mouse MDR3 P-glycoprotein (Pgp) was constructed by mutagenesis of the nine natural Cys to Ala. The Cys-free protein was expressed in Pichia pastoris and purified. Yield, purity, ATPase activity, K(m)(MgATP), and stimulation of ATPase by verapamil, were similar to wild-type mouse Ppg. Mouse Cys-free Pgp was superior in yield and stability to Cys-free human MDR1 Pgp. Mutants Y1040A and Y1040C were constructed in mouse Cys-free Pgp background. Both showed extremely low ATPase activity, strongly-impaired vanadate-trapping of ADP, and reduced photolabeling by 8-azido-ATP. The results are consistent with the conclusion that Tyr-1040 is located in the MgATP-binding site in NBD2 and is required for correct binding and/or orientation of bound MgATP substrate in Pgp as previously suggested by X-ray structures of other ABC transporters and by sequencing of photolabeled Pgp. The results also support our previous conclusion that both catalytic sites must be intact for normal function in Pgp.

Nucleotide binding to the multidrug resistance P-glycoprotein as studied by ESR spectroscopy.

Electron spin resonance (ESR) spectroscopy using spin-labeled ATP was used to study nucleotide binding to and structural transitions within the multidrug resistance P-glycoprotein, P-gp. Spin-labeled ATP (SL-ATP) with the spin label attached to the ribose, was observed to be an excellent substrate analogue for P-gp. SL-ATP was hydrolyzed in a drug-stimulated fashion at about 14% of the rate for normal ATP and allowed reversible trapping of the enzyme in transition and ground states. Equilibrium binding of a total of two nucleotides per P-gp was observed with a binding affinity of 366 microM in the presence of Mg2+ but in the absence of transport substrates such as verapamil. Binding of SL-ATP to wild-type P-gp in the presence of verapamil resulted in reduction of the protein-bound spin-label moiety, most likely due to a conformational transition within P-gp that positioned cysteines in close proximity to the spin label to allow chemical reduction of the radical. We circumvented this problem by using a mutant of P-gp in which all naturally occurring cysteines were substituted for alanines. Equilibrium binding of SL-ATP to this mutant P-gp resulted in maximum binding of two nucleotides; the binding affinity was 223 microM in the absence and 180 microM in the presence of verapamil. The corresponding ESR spectra of wild-type and Cys-less P-gp in the presence of SL-ATP indicate that a cysteine side chain of P-gp is located close to the ribose of the bound nucleotide. Trapping SL-ATP as an AlF(x)-adduct resulted in ESR spectra that showed strong immobilization of the radical, supporting the formation of a closed conformation of P-gp in its transition state. This study is the first to employ ESR spectroscopy with the use of spin-labeled nucleotide analogues to study P-glycoprotein. The study shows that SL-ATP is an excellent substrate analogue that will allow further exploration of structure and dynamics within the nucleotide binding domains of this important enzyme.

Involvement of the "occluded nucleotide conformation" of P-glycoprotein in the catalytic pathway.

We found recently that the combined mutation of both "catalytic carboxylate" residues (E552A/E1197A) in mouse P-glycoprotein (Pgp) arrested the protein in an "occluded nucleotide conformation", possibly a stabilized dimer of nucleotide-binding domains (NBDs), that binds MgATP tightly at stoichiometry of 1 mol/mol Pgp [Tombline, G., Bartholomew, L., Urbatsch, I. L., and Senior, A. E. (2004) J. Biol. Chem. 279, 31212-31220]. Here, we further examine this conformation in respect to its potential involvement in the catalytic pathway. The occluded nucleotide conformation is promoted by drugs. Verapamil markedly accelerated the rate of tight binding of MgATP, whereas it did not effect the rate of dissociation. Mutations in "Q-loop" residues that are thought to interfere with communication between drug and catalytic sites prevented the occluded nucleotide conformation, as did covalent reagents N-ethylmaleimide and 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole, which are known to inhibit ATP hydrolysis by reacting in catalytic sites. Mutations of Walker A Ser and Lys residues in combination with E552A/E1197A had the same effect, showing that interaction of these conserved residues with MgATP is required to stabilize the occluded nucleotide conformation. We present an enzymatic scheme that incorporates this conformation. We propose that upon initial loose binding of MgATP at two nucleotide-binding domains (NBDs), together with drug binding, the NBDs dimerize to form the occluded conformation, with one tightly bound MgATP committed to hydrolysis. The pathway progresses such that the tightly bound MgATP enters the transition state and is hydrolyzed. This work suggests that small molecules or peptides that interact at the NBD dimer interface might effectively disable Pgp catalysis.

Structural characterization of the interaction of the delta and alpha subunits of the Escherichia coli F1F0-ATP synthase by NMR spectroscopy.

A critical point of interaction between F(1) and F(0) in the bacterial F(1)F(0)-ATP synthase is formed by the alpha and delta subunits. Previous work has shown that the N-terminal domain (residues 3-105) of the delta subunit forms a 6 alpha-helix bundle [Wilkens, S., Dunn, S. D., Chandler, J., Dahlquist, F. W., and Capaldi, R. A. (1997) Nat. Struct. Biol. 4, 198-201] and that the majority of the binding energy between delta and F(1) is provided by the interaction between the N-terminal 22 residues of the alpha- and N-terminal domain of the delta subunit [Weber, J., Muharemagic, A., Wilke-Mounts, S., and Senior, A. E. (2003) J. Biol. Chem. 278, 13623-13626]. We have now analyzed a 1:1 complex of the delta-subunit N-terminal domain and a peptide comprising the N-terminal 22 residues of the alpha subunit by heteronuclear protein NMR spectroscopy. A comparison of the chemical-shift values of delta-subunit residues with and without alpha N-terminal peptide bound indicates that the binding interface on the N-terminal domain of the delta subunit is formed by alpha helices I and V. NOE cross-peak patterns in 2D (12)C/(12)C-filtered NOESY spectra of the (13)C-labeled delta-subunit N-terminal domain in complex with unlabeled peptide verify that residues 8-18 in the alpha-subunit N-terminal peptide are folded as an alpha helix when bound to delta N-terminal domain. On the basis of intermolecular contacts observed in (12)C/(13)C-filtered NOESY experiments, we describe structural details of the interaction of the delta-subunit N-terminal domain with the alpha-subunit N-terminal alpha helix.

Modulation of charge in the phosphate binding site of Escherichia coli ATP synthase.

This paper presents a study of the role of positive charge in the P(i) binding site of Escherichia coli ATP synthase, the enzyme responsible for ATP-driven proton extrusion and ATP synthesis by oxidative phosphorylation. Arginine residues are known to occur with high propensity in P(i) binding sites of proteins generally and in the P(i) binding site of the betaE catalytic site of ATP synthase specifically. Removal of natural betaArg-246 (betaR246A mutant) abrogates P(i) binding; restoration of P(i) binding was achieved by mutagenesis of either residue betaAsn-243 or alphaPhe-291 to Arg. Both residues are located in the P(i) binding site close to betaArg-246 in x-ray structures. Insertion of one extra Arg at beta-243 or alpha-291 in presence of betaArg-246 retained P(i) binding, but insertion of two extra Arg, at both positions simultaneously, abrogated it. Transition state stabilization was measured using phosphate analogs fluoroaluminate and fluoroscandium. Removal of betaArg-246 in betaR246A caused almost complete loss of transition state stabilization, but partial rescue was achieved in betaN243R/betaR246A and alphaF291R/betaR246A. BetaArg-243 or alphaArg-291 in presence of betaArg-246 was less effective; the combination of alphaF291R/betaN243R with natural betaArg-246 was just as detrimental as betaR246A. The data demonstrate that electrostatic interaction is an important component of initial P(i) binding in catalytic site betaE and later at the transition state complex. However, since none of the mutants showed significant function in growth tests, ATP-driven proton pumping, or ATPase activity assays, it is apparent that specific stereochemical interactions of catalytic site Arg residues are paramount.

Involvement of ATP synthase residues alphaArg-376, betaArg-182, and betaLys-155 in Pi binding.

alphaArg-376, betaLys-155, and betaArg-182 are catalytically important ATP synthase residues that were proposed to be involved in substrate Pi binding and subsequent steps of ATP synthesis [Senior, A.E., Nadanaciva, S. and Weber, J. (2002) Biochim. Biophys. Acta 1553, 188-211]. Here, it was shown using purified Escherichia coli F(1)-ATPase that whereas Pi protected wild-type from reaction with 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole, mutations betaK155Q, betaR182Q, betaR182K, and alphaR376Q abolished protection. Therefore, in ATP synthesis initial binding of substrate Pi in open catalytic site betaE is supported by each of these three residues.

Identification of phosphate binding residues of Escherichia coli ATP synthase.

Four positively-charged residues, namely betaLys-155, betaArg-182, betaArg-246, and alphaArg-376 have been identified as Pi binding residues in Escherichia coli ATP synthase. They form a triangular Pi binding site in catalytic site betaE where substrate Pi initially binds for ATP synthesis in oxidative phosphorylation. Positive electrostatic charge in the vicinity of betaArg-246 is shown to be one important component of Pi binding.

The occluded nucleotide conformation of p-glycoprotein.

We review recent work on E552A/E1197A P-glycoprotein. This ATPase-defective mutant occludes MgATP tightly with maximal 1/1 stoichiometry in drug-sensitive fashion. The occluded nucleotide conformation appears to represent a transient, asymmetric, catalytic intermediate. We present a model for catalysis incorporating nucleotide binding domain (NBD) dimerization and the occluded nucleotide conformation, and we speculate as to how catalysis seen in P-glycoprotein might be harmonized with symmetrical dimer structures of isolated NBDs.

Properties of P-glycoprotein with mutations in the "catalytic carboxylate" glutamate residues.

It is known from earlier work that two conserved Glu residues, designated "catalytic carboxylates," are critical for function in P-glycoprotein (Pgp). Here the role of these residues (Glu-552 and Glu-1197 in mouse MDR3 Pgp) was studied further. Mutation E552Q or E1197Q reduced Pgp-ATPase to low but still measurable rates. Two explanations previously offered for effects of these mutations, namely that ADP release is slowed or that a second (drug site-resetting) round of ATP hydrolysis is blocked, were evaluated and appeared unsatisfactory. Thus the study was extended to include E552A, -D, and -K and E1197A, -D, and -K mutants. All reduced ATPase to similar low but measurable rates. Orthovanadate-trapping experiments showed that mutation to Gln, Ala, Asp, or Lys altered characteristics of the transition state but did not eliminate its formation in contrast e.g. with mutation of the analogous catalytic Glu in F1-ATPase. Retention of ATP as well as ADP was seen in Ala, Asp, and Lys mutants. Mutation E552A in nucleotide binding domain 1 (NBD1) was combined with mutation S528A or S1173A in the LSGGQ sequence of NBD1 or NBD2, respectively. Synergistic effects were seen. E552A/S1173A had extremely low turnover rate for ATPase, while E552A/S528A showed zero or close to zero ATPase. Both showed orthovanadate-independent retention of ATP and ADP. We propose that mutations of the catalytic Glu residues interfere with formation and characteristics of a closed conformation, involving an interdigitated NBD dimer interface, which normally occurs immediately following ATP binding and progresses to the transition state.

Role of betaAsn-243 in the phosphate-binding subdomain of catalytic sites of Escherichia coli F(1)-ATPase.

In the catalytic mechanism of ATP synthase, phosphate (P(i)) binding and release steps are believed to be correlated to gamma-subunit rotation, and P(i) binding is proposed to be prerequisite for binding ADP in the face of high cellular [ATP]/[ADP] ratios. In x-ray structures, residue betaAsn-243 appears centrally located in the P(i)-binding subdomain of catalytic sites. Here we studied the role of betaAsn-243 in Escherichia coli ATP synthase by mutagenesis to Ala and Asp. Mutation betaN243A caused 30-fold impairment of F(1)-ATPase activity; 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole inhibited this activity less potently than in wild type and P(i) protected from inhibition. ADP-fluoroaluminate was more inhibitory than in wild-type, but ADP-fluoroscandium was less inhibitory. betaN243D F(1)-ATPase activity was impaired by 1300-fold and was not inhibited by ADP-fluoroaluminate or ADP-fluoroscandium. 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole activated betaN243D F(1)-ATPase, and P(i) did not affect activation. We conclude that residue betaAsn-243 is not involved in P(i) binding directly but is necessary for correct organization of the transition state complex through extensive involvement in hydrogen bonding to neighboring residues. It is also probably involved in orientation of the "attacking water" and of an associated second water.

Mutagenesis of residue betaArg-246 in the phosphate-binding subdomain of catalytic sites of Escherichia coli F1-ATPase.

Residues responsible for phosphate binding in F(1)F(0)-ATP synthase catalytic sites are of significant interest because phosphate binding is believed linked to proton gradient-driven subunit rotation. From x-ray structures, a phosphate-binding subdomain is evident in catalytic sites, with conserved betaArg-246 in a suitable position to bind phosphate. Mutations betaR246Q, betaR246K, and betaR246A in Escherichia coli were found to impair oxidative phosphorylation and to reduce ATPase activity of purified F(1) by 100-fold. In contrast to wild type, ATPase of mutants was not inhibited by MgADP-fluoroaluminate or MgADP-fluoroscandium, showing the Arg side chain is required for wild-type transition state formation. Whereas 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole (NBD-Cl) inhibited wild-type ATPase essentially completely, ATPase in mutants was inhibited maximally by approximately 50%, although reaction still occurred at residue betaTyr-297, proximal to betaArg-246 in the phosphate-binding pocket. Inhibition characteristics supported the conclusion that NBD-Cl reacts in betaE (empty) catalytic sites, as shown previously by x-ray structure analysis. Phosphate protected against NBD-Cl inhibition in wild type but not in mutants. The results show that phosphate can bind in the betaE catalytic site of E. coli F(1) and that betaArg-246 is an important phosphate-binding residue.

Combined mutation of catalytic glutamate residues in the two nucleotide binding domains of P-glycoprotein generates a conformation that binds ATP and ADP tightly.

Combined mutation of "catalytic carboxylates" in both nucleotide binding domains (NBDs) of P-glycoprotein generates a conformation capable of tight binding of 8-azido-ADP (Sauna, Z. E., Müller, M., Peng, X. H., and Ambudkar, S. V. (2002) Biochemistry 41, 13989-14000). Here we characterized this conformation using pure mouse MDR3 P-glycoprotein and natural MgATP and MgADP. Mutants E552A/E1197A, E552Q/E1197Q, E552D/E1197D, and E552K/E1197K had low but real ATPase activity in the order Ala > Gln > Asp > Lys, emphasizing the requirement for Glu stereochemistry. Mutant E552A/E1197A bound MgATP and MgADP (1 mol/mol) with K(d) 9.2 and 92 microm, showed strong temperature sensitivity of MgATP binding and equal dissociation rates for MgATP and MgADP. With MgATP as the added ligand, 80% of bound nucleotide was in the form of ATP. None of these parameters was vanadate-sensitive. The other mutants showed lower stoichiometry of MgATP and MgADP binding, in the order Ala > Gln > Asp > Lys. We conclude that the E552A/E1197A mutation arrests the enzyme in a conformation, likely a stabilized NBD dimer, which occludes nucleotide, shows preferential binding of ATP, does not progress to a normal vanadate-sensitive transition state, but hydrolyzes ATP and releases ADP slowly. Impairment of turnover is primarily due to inability to form the normal transition state rather than to slow ADP release. The Gln, Asp, and Lys mutants are less effective at stabilizing the occluded nucleotide, putative dimeric NBD, conformation. We envisage that in wild-type the occluded nucleotide conformation occurs transiently after MgATP binds to both NBDs with associated dimerization, and before progression to the transition state.