Expression, Isolation, and Crystallization of the Catalytic Domain of CopB, a Putative Copper Transporting ATPase from the Thermoacidophilic Archaeon Sulfolobus solfataricus

The P-type CPX-ATPases are responsible for the transport of heavy metal ions in archaea, bacteria, and eukaryotes. We have chosen one of the two CPX-ATPases of the thermophile Sulfolobus solfataricus, CopB (= SSO2896) for the investigation of the molecular mechanism of this integral membrane protein. We recombinately expressed three different soluble domains of this protein (named CopB-A, CopB-B, and CopB-C) in Escherichia coli and purified them to homogeneity. 3D crystals of CopB-B, the 29 kDa catalytic ATP binding/phosphorylation domain were produced, which diffracted to a resolution of 2.2 A. CopB-B has heavy metal stimulated phosphatase activity, which was half maximal in the presence of 80 microM Cu2+. The protein forms a phosphorylated intermediate with the substrate gamma-(32P)-ATP. No specific activation of the polypeptide was observed, when CopB-B phosphatase activity was tested in the presence of the purified CopB-C and CopB-A proteins, which provide the cation binding and the phosphatase domains. We conclude that CopB is a putatively copper translocating ATPase, in which structural elements integrally located in the membrane are required for full, coordinated activation of the catalytic ATP binding domain.     

Introducing Wilson disease mutations into the zinc-transporting P-type ATPase of Escherichia coli. The mutation P634L in the 'hinge' motif (GDGXNDXP) perturbs the formation of the E2P state.

ZntA, a bacterial zinc-transporting P-type ATPase, is homologous to two human ATPases mutated in Menkes and Wilson diseases. To explore the roles of the bacterial ATPase residues homologous to those involved in the human diseases, we have introduced several point mutations into ZntA. The mutants P401L, D628A and P634L correspond to the Wilson disease mutations P992L, D1267A and P1273L, respectively. The mutations D628A and P634L are located in the C-terminal part of the phosphorylation domain in the so-called hinge motif conserved in all P-type ATPases. P401L resides near the N-terminal portion of the phosphorylation domain whereas the mutations H475Q and P476L affect the heavy metal ATPase-specific HP motif in the nucleotide binding domain. All mutants show reduced ATPase activity corresponding 0-37% of the wild-type activity. The mutants P401L, H475Q and P476L are poorly phosphorylated by both ATP and P(i). Their dephosphorylation rates are slow. The D628A mutant is inactive and cannot be phosphorylated at all. In contrast, the mutant P634L six residues apart in the same domain shows normal phosphorylation by ATP. However, phosphorylation by P(i) is almost absent. In the absence of added ADP the P634L mutant dephosphorylates much more slowly than the wild-type, whereas in the presence of ADP the dephosphorylation rate is faster than that of the wild-type. We conclude that the mutation P634L affects the conversion between the states E1P and E2P so that the mutant favors the E1 or E1P state.     

The holo-form of the nucleotide binding domain of the KdpFABC complex from Escherichia coli reveals a new binding mode

P-type ATPases are ubiquitously abundant enzymes involved in active transport of charged residues across biological membranes. The KdpB subunit of the prokaryotic Kdp-ATPase (KdpFABC complex) shares characteristic regions of homology with class II-IV P-type ATPases and has been shown previously to be misgrouped as a class IA P-type ATPase. Here, we present the NMR structure of the AMP-PNP-bound nucleotide binding domain KdpBN of the Escherichia coli Kdp-ATPase at high resolution. The aromatic moiety of the nucleotide is clipped into the binding pocket by Phe(377) and Lys(395) via a pi-pi stacking and a cation-pi interaction, respectively. Charged residues at the outer rim of the binding pocket (Arg(317), Arg(382), Asp(399), and Glu(348)) stabilize and direct the triphosphate group via electrostatic attraction and repulsion toward the phosphorylation domain. The nucleotide binding mode was corroborated by the replacement of critical residues. The conservative mutation F377Y produced a high residual nucleotide binding capacity, whereas replacement by alanine resulted in low nucleotide binding capacities and a considerable loss of ATPase activity. Similarly, mutation K395A resulted in loss of ATPase activity and nucleotide binding affinity, even though the protein was properly folded. We present a schematic model of the nucleotide binding mode that allows for both high selectivity and a low nucleotide binding constant, necessary for the fast and effective turnover rate realized in the reaction cycle of the Kdp-ATPase.     

Prokaryotic Kdp-ATPase: recent insights into the structure and function of KdpB

P-type ATPases are amongst the most abundant enzymes that are responsible for active transport of ions across biological membranes. Within the last 5 years a detailed picture of the structure and function of these transport ATPases has emerged. Here, we report on the recent progress in elucidating the molecular mechanism of a unique, prokaryotic member of P-type ATPases, the Kdp-ATPase. The review focuses on the catalytic parts of the central subunit, KdpB. The structure of the nucleotide-binding domain was solved by NMR spectroscopy at high resolution and a model of the nucleotide-binding mode was presented. The nucleotide turned out to be 'clipped' into the binding pocket by a pi-pi interaction to F377 on one side and a cation-pi interaction to K395 on the other. The 395KGXXD/E motif and thus the nucleotide-binding mode seems to be conserved in all P-type ATPases, except the heavy metal-transporting (class IB) ATPases. Hence, it can be concluded that KdpB is currently misgrouped as class IA. Mutational studies on two highly conserved residues (D583 and K586) in the transmembrane helix 5 of KdpB revealed that they are indispensable in coupling ATP hydrolysis to ion translocation. Based on these results, two possible pathways for the reaction cycle are discussed.     

Effect of orthophosphate, nucleotide analogues, ADP, and phosphorylation on the cytoplasmic domains of Ca(2+)-ATPase from scallop sarcoplasmic reticulum

The effects of orthophosphate, nucleotide analogues, ADP, and covalent phosphorylation on the tryptic fragmentation patterns of the E1 and E2 forms of scallop Ca-ATPase were examined. Sites preferentially cleaved by trypsin in the E1 form of the Ca-ATPase were detected in the nucleotide (N) and phosphorylation (P) domains, as well as the actuator (A) domain. These sites were occluded in the E2 (Ca(2+)-free) form of the enzyme, consistent with mutual protection of the A, N, and P domains through their association into a clustered structure. Similar protection of cytoplasmic Ca(2+)-dependent tryptic cleavage sites was observed when the catalytic binding site for substrate on the E1 form of scallop Ca-ATPase was occupied by Pi, AMP-PNP, AMP-PCP, or ADP despite the presence of saturating levels of Ca2+. These results suggest that occupation of the catalytic site on E1 can induce condensation of the cytoplasmic domains to yield a unique structural intermediate that may be related to the form of the enzyme in which the active site is prepared for phosphoryl transfer. The effect of Pi on the E2 form of the scallop Ca-ATPase was also investigated, when it was found that formation of E2-P led to extreme resistance toward secondary cleavage by trypsin and stabilization of enzymatic activity for long periods of time.     

Crystal structure of phosphoserine phosphatase from Methanococcus jannaschii, a hyperthermophile, at 1.8 A resolution

BACKGROUND: D-Serine is a co-agonist of the N-methyl-D-aspartate subtype of glutamate receptors, a major neurotransmitter receptor family in mammalian nervous systems. D-Serine is converted from L-serine, 90% of which is the product of the enzyme phosphoserine phosphatase (PSP). PSP from M. jannaschii (MJ) shares significant sequence homology with human PSP. PSPs and P-type ATPases are members of the haloacid dehalogenase (HAD)-like hydrolase family, and all members share three conserved sequence motifs. PSP and P-type ATPases utilize a common mechanism that involves Mg(2+)-dependent phosphorylation and autodephosphorylation at an aspartyl side chain in the active site. The strong resemblance in sequence and mechanism implies structural similarity among these enzymes. RESULTS: The PSP crystal structure resembles the NAD(P) binding Rossmann fold with a large insertion of a four-helix-bundle domain and a beta hairpin. Three known conserved sequence motifs are arranged next to each other in space and outline the active site. A phosphate and a magnesium ion are bound to the active site. The active site is within a closed environment between the core alpha/beta domain and the four-helix-bundle domain. CONCLUSIONS: The crystal structure of MJ PSP was determined at 1.8 A resolution. Critical residues were assigned based on the active site structure and ligand binding geometry. The PSP structure is in a closed conformation that may resemble the phosphoserine bound state or the state after autodephosphorylation. Compared to a P-type ATPase (Ca(2+)-ATPase) structure, which is in an open state, this PSP structure appears also to be a good model for the closed conformation of P-type ATPase.     

Metal fluoride complexes of Na,K-ATPase: characterization of fluoride-stabilized phosphoenzyme analogues and their interaction with cardiotonic steroids

The Na,K-ATPase belongs to the P-type ATPase family of primary active cation pumps. Metal fluorides like magnesium-, beryllium-, and aluminum fluoride act as phosphate analogues and inhibit P-type ATPases by interacting with the phosphorylation site, stabilizing conformations that are analogous to specific phosphoenzyme intermediates. Cardiotonic steroids like ouabain used in the treatment of congestive heart failure and arrhythmias specifically inhibit the Na,K-ATPase, and the detailed structure of the highly conserved binding site has recently been described by the crystal structure of the shark Na,K-ATPase in a state analogous to E2·2K(+)·P(i) with ouabain bound with apparently low affinity (1). In the present work inhibition, and subsequent reactivation by high Na(+), after treatment of shark Na,K-ATPase with various metal fluorides are characterized. Half-maximal inhibition of Na,K-ATPase activity by metal fluorides is in the micromolar range. The binding of cardiotonic steroids to the metal fluoride-stabilized enzyme forms was investigated using the fluorescent ouabain derivative 9-anthroyl ouabain and compared with binding to phosphorylated enzyme. The fastest binding was to the Be-fluoride stabilized enzyme suggesting a preformed ouabain binding cavity, in accord with results for Ca-ATPase where Be-fluoride stabilizes the E2-P ground state with an open luminal ion access pathway, which in Na,K-ATPase could be a passage for ouabain. The Be-fluoride stabilized enzyme conformation closely resembles the E2-P ground state according to proteinase K cleavage. Ouabain, but not its aglycone ouabagenin, prevented reactivation of this metal fluoride form by high Na(+) demonstrating the pivotal role of the sugar moiety in closing the extracellular cation pathway.     

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.     

The ATPase mechanism of ArsA, the catalytic subunit of the arsenite pump.

The ArsA ATPase is the catalytic subunit of a novel arsenite pump, with two nucleotide-binding consensus sequences in the N- and C-terminal halves of the protein. The single tryptophan-containing Trp159 ArsA was used to elucidate the elementary steps of the ATPase mechanism by fluorescence stopped-flow experiments. The binding and hydrolysis of MgATP is a multistep process with a minimal kinetic mechanism (Mechanism 1). A notable feature of the reaction is that MgATP binding induces a slow transient increase in fluorescence of ArsA, which is independent of the ATP concentration, indicative of the build-up of a pre-steady state intermediate. This finding, coupled with a phosphate burst, implies that the steady-state intermediate builds up subsequent to product release. We propose that the rate-limiting step is an isomerization between different conformational forms of ArsA. kcat is faster than the phosphate burst, indicating that both nucleotide binding sites of ArsA are catalytic. Consistent with this interpretation, approximately 2 mol of phosphate are released per mole of ArsA during the phosphate burst.     

Functional modules of KdpB, the catalytic subunit of the Kdp-ATPase from Escherichia coli

The large cytoplasmic domain (H4H5) of KdpB of the KdpFABC complex (P-type ATPase) from Escherichia coli consists of two separate modules, the phosphorylation domain (KdpBP) and the nucleotide binding domain (KdpBN). The H4H5 and the KdpBN domains were purified as soluble 10His-tagged fusion proteins. Both proteins exhibit a mainly alpha-helical secondary structure as judged by CD spectroscopy. Fluorescein 5-isothiocyanate (FITC) labeling studies revealed that both proteins form a proper nucleotide binding site. Adenosine nucleotides protect the H4H5 loop but not KdpBN against FITC modification. Trinitrophenyl (TNP)-nucleotide binding studies revealed that both H4H5 and KdpBN bind nucleotides with high affinity. Furthermore, the H4H5 loop was still able to hydrolyze ATP, as well as p-nitrophenyl phosphate (pNPP). These results lend support to the notion that the separately synthesized H4H5 and KdpBN domains retain their native structure and that they reveal properties of both P2-type ATPases (e.g., Na(+),K(+)-ATPase and Ca(2+)-ATPase) and P1b-type ATPases (e.g., heavy metal transporting ATPases). Furthermore, this report also emphasizes the unique position of the Kdp-ATPase within the P-type ATPase family.     

Conformational changes in sarcoplasmic reticulum Ca(2+)-ATPase mutants: effect of mutations either at Ca(2+)-binding site II or at tryptophan 552 in the cytosolic domain

By analyzing, after expression in yeast and purification, the intrinsic fluorescence properties of point mutants of rabbit Ca(2+)-ATPase (SERCA1a) with alterations to amino acid residues in Ca(2+)-binding site I (E(771)), site II (E(309)), in both sites (D(800)), or in the nucleotide-binding domain (W(552)), we were able to follow the conformational changes associated with various steps in the ATPase catalytic cycle. Whereas Ca(2+) binding to purified wild-type (WT) ATPase in the absence of ATP leads to the rise in Trp fluorescence expected for the so-called E2 --> E1Ca(2) transition, the Ca(2+)-induced fluorescence rise is dramatically reduced for the E(309)Q mutant. As this purified E(309)Q mutant retains the ability to bind Ca(2+) at site I (but not at site II), we tentatively conclude that the protein reorganization induced by Ca(2+) binding at site II makes the major contribution to the overall Trp fluorescence changes observed upon Ca(2+) binding to both sites. Judging from the fluorescence response of W(552)F, similar to that of WT, these changes appear to be primarily due to membranous tryptophans, not to W(552). The same holds for the fluorescence rise observed upon phosphorylation from P(i) (the so-called E2 --> E2P transition). As for WT ATPase, Mg(2+) binding in the absence of Ca(2+) affects the fluorescence of the E(309)Q mutant, suggesting that this Mg(2+)-dependent fluorescence rise does not reflect binding of Mg(2+) to Ca(2+) sites; instead, Mg(2+) probably binds close to the catalytic site, or perhaps near transmembrane span M3, at a location recently revealed by Fe(2+)-catalyzed oxidative cleavage. Mutation of W(552) hardly affects ATP-induced fluorescence changes in the absence of Ca(2+), which are therefore mostly due to membranous Trp residues, demonstrating long-range communication between the nucleotide-binding domain and the membranous domain.     

Crystal structure of dephospho-coenzyme A kinase from Haemophilus influenzae

Dephospho-coenzyme A kinase catalyzes the final step in CoA biosynthesis, the phosphorylation of the 3'-hydroxyl group of ribose using ATP as a phosphate donor. The protein from Haemophilus influenzae was cloned and expressed, and its crystal structure was determined at 2.0-A resolution in complex with ATP. The protein molecule consists of three domains: the canonical nucleotide-binding domain with a five-stranded parallel beta-sheet, the substrate-binding alpha-helical domain, and the lid domain formed by a pair of alpha-helices. The overall topology of the protein resembles the structures of nucleotide kinases. ATP binds in the P-loop in a manner observed in other kinases. The CoA-binding site is located at the interface of all three domains. The double-pocket structure of the substrate-binding site is unusual for nucleotide kinases. Amino acid residues implicated in substrate binding and catalysis have been identified. The structure analysis suggests large domain movements during the catalytic cycle.     

Calcium activation of the Ca-ATPase enhances conformational heterogeneity between nucleotide binding and phosphorylation domains

High-resolution crystal structures obtained in two conformations of the Ca-ATPase suggest that a large-scale rigid-body domain reorientation of approximately 50 degrees involving the nucleotide-binding (N) domain is required to permit the transfer of the gamma-phosphoryl group of ATP to Asp(351) in the phosphorylation (P) domain during coupled calcium transport. However, variability observed in the orientations of the N domain relative to the P domain in the different crystal structures of the Ca-ATPase following calcium activation and the structures of other P-type ATPases suggests the presence of conformational heterogeneity in solution, which may be modulated by contact interactions within the crystal. Therefore, to address the extent of conformational heterogeneity between these domains in solution, we have used fluorescence resonance energy transfer to measure the spatial separation and conformational heterogeneity between donor (i.e., 5-[[2-[(iodoacetyl)amino]ethyl]amino]naphthalene-1-sulfonic acid) and acceptor (i.e., fluorescein 5-isothiocyanate) chromophores covalently bound to the P and N domains, respectively, within the Ca-ATPase stabilized in different enzymatic states associated with the transport cycle. In comparison to the unliganded enzyme, the spatial separation and conformational heterogeneity between these domains are unaffected by enzyme phosphorylation. However, calcium activation results in a 3.4 A increase in the average spatial separation, from 29.4 to 32.8 A, in good agreement with the 4.3 A increase in the distance estimated from high-resolution structures where these sites are respectively separated by 31.6 A (1IWO.pdb) and 35.9 A (1EUL.pdb). Thus, the crystal structures accurately reflect the average solution structures of the Ca-ATPase. These results suggest that the approximation of cytoplasmic domains accompanying calcium transport, as observed from crystal structures, occurs in solution within the context of large amplitude domain motions important for catalysis. We suggest that these domain motions enhance the rates of substrate (ATP) access and product (ADP) egress and the probability of a productive juxtaposition of the gamma-phosphoryl moiety of ATP with Asp(351) on the phosphorylation domain to facilitate enzyme phosphorylation and calcium transport.     

The regulation of catalytic activity of the menkes copper-translocating P-type ATPase. Role of high affinity copper-binding sites

The Menkes protein is a transmembrane copper translocating P-type ATPase. Mutations in the Menkes gene that affect the function of the Menkes protein may cause Menkes disease in humans, which is associated with severe systemic copper deficiency. The catalytic mechanism of the Menkes protein, including the formation of transient acylphosphate, is poorly understood. We transfected and overexpressed wild-type and targeted mutant Menkes protein in yeast and investigated its transient acyl phosphorylation. We demonstrated that the Menkes protein is transiently phosphorylated by ATP in a copper-specific and copper-dependent manner and appears to undergo conformational changes in accordance with the classical P-type ATPase model. Our data suggest that the catalytic cycle of the Menkes protein begins with the binding of copper to high affinity binding sites in the transmembrane channel, followed by ATP binding and transient phosphorylation. We propose that putative copper-binding sites at the N-terminal domain of the Menkes protein are important as sensors of low concentrations of copper but are not essential for the overall catalytic activity.     

Structure of dimeric SecA, the Escherichia coli preprotein translocase motor

SecA is the preprotein translocase ATPase subunit and a superfamily 2 (SF2) RNA helicase. Here we present the 2 A crystal structures of the Escherichia coli SecA homodimer in the apo form and in complex with ATP, ADP and adenosine 5'-[beta,gamma-imido]triphosphate (AMP-PNP). Each monomer contains the SF2 ATPase core (DEAD motor) built of two domains (nucleotide binding domain, NBD and intramolecular regulator of ATPase 2, IRA2), the preprotein binding domain (PBD), which is inserted in NBD and a carboxy-terminal domain (C-domain) linked to IRA2. The structures of the nucleotide complexes of SecA identify an interfacial nucleotide-binding cleft located between the two DEAD motor domains and residues critical for ATP catalysis. The dimer comprises two virtually identical protomers associating in an antiparallel fashion. Dimerization is mediated solely through extensive contacts of the DEAD motor domains leaving the C-domain facing outwards from the dimerization core. This dimerization mode explains the effect of functionally important mutations and is completely different from the dimerization models proposed for other SecA structures. The repercussion of these findings on translocase assembly and catalysis is discussed.     

ATP-induced conformational changes of the nucleotide-binding domain of Na,K-ATPase

The Na,K-ATPase hydrolyzes ATP to drive the coupled extrusion and uptake of Na+ and K+ ions across the plasma membrane. Here, we report two high-resolution NMR structures of the 213-residue nucleotide-binding domain of rat alpha1 Na,K-ATPase, determined in the absence and the presence of ATP. The nucleotide binds in the anti conformation and shows a relative paucity of interactions with the protein, reflecting the low-affinity ATP-binding state. Binding of ATP induces substantial conformational changes in the binding pocket and in residues located in the hinge region connecting the N- and P-domains. Structural comparison with the Ca-ATPase stabilized by the inhibitor thapsigargin, E2(TG), and the model of the H-ATPase in the E1 form suggests that the observed changes may trigger the series of events necessary for the release of the K+ ions and/or disengagement of the A-domain, leading to the eventual transfer of the gamma-phosphate group to the invariant Asp369.     

Rearrangement of domain elements of the Ca-ATPase in cardiac sarcoplasmic reticulum membranes upon phospholamban phosphorylation.

Phospholamban (PLB) is a major target of the beta-adrenergic cascade in the heart, and functions to modulate rate-limiting conformational transitions involving the transport activity of the Ca-ATPase. To investigate structural changes within the Ca-ATPase that result from the phosphorylation of PLB by cAMP-dependent protein kinase (PKA), we have covalently bound the long-lived phosphorescent probe erythrosin isothiocyanate (Er-ITC) to cytoplasmic sequences within the Ca-ATPase. Under these labeling conditions, the Ca-ATPase remains catalytically active, indicating that observed changes in rotational dynamics reflect normal conformational transitions. Two major Er-ITC labeling sites were identified using electrospray ionization mass spectrometry (ESI-MS), corresponding to Lys464 and Lys650, which are respectively located within the phosphorylation and nucleotide binding domains of the Ca-ATPase. Frequency-domain phosphorescence measurements of the rotational dynamics of Er-ITC bound to these cytoplasmic sequences within the Ca-ATPase permit the resolution of the dynamic structure of individual domain elements relative to the overall rotational motion of the entire Ca-ATPase polypeptide chain. We observe a significant decrease in the rotational dynamics of Er-ITC bound to the Ca-ATPase upon phosphorylation of PLB by PKA, as evidenced by an increase in the residual anisotropy. These results suggest that phosphorylation of PLB results in a structural reorientation of the phosphorylation or nucleotide binding domains with respect to the membrane normal. In contrast, calcium activation of the Ca-ATPase in the presence of dephosphorylated PLB results in no detectable change in the rotational dynamics of Er-ITC, suggesting that calcium binding and PLB phosphorylation have distinct effects on the conformation of the Ca-ATPase. We suggest that PLB functions to alter the efficiency of phosphoenyzme formation following calcium activation of the Ca-ATPase by modulating the spatial arrangement between ATP bound in the nucleotide binding domain and Asp351 in the phosphorylation domain.     

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.     

Functional and structural roles of critical amino acids within the"N", "P", and "A" domains of the Ca2+ ATPase (SERCA) headpiece

Twenty five amino acids within the "N", "P", and "A" domains of the Ca(2+) ATPase (SERCA1) headpiece were subjected to site directed mutagenesis, taking advantage of a high yield expression system. Functional and conformational effects of mutations were interpreted systematically in the light of the high resolution WT structure, defining direct involvement in catalysis as well as in stabilization of various positions acquired by each domain upon substrate binding and utilization. Amino acids involved in binding of ATP (such as Phe487 and Arg560 in the N domain) or phosphate (such as Asp351, Thr625, Lys684, and Thr353 in the P domain) were characterized with respect to their binding mechanism. Further identified were "positional" roles of several amino acids that stabilize neighboring residues for optimal binding of substrate or Mg(2+), or interface between headpiece domains as they change their relative positions in the course of the catalytic cycle. These include cross-linking of the "N" and "P" domains (e.g., Arg560/Asp627 salt bridge to stabilize domain approximation by ATP binding), and stabilization of the "A", "N", and activated "P" domains in arrangements differing from the ground E2 state and driven by catalytic events. This stabilization is produced through hydrogen bonds at domain interfaces, which vary depending on the intermediate state (e.g., Glu486/T171 in E1P and E2P, as opposed to Glu486/H190 in E2). We demonstrate that specific arrangements of the headpiece domains shown in crystal structures are, in fact, required to trigger displacement of transmembrane segments during the enzyme cycle in solution, allowing long range linkage of catalytic and Ca(2+) binding functions.