Binding Of AMP-PNP Research Articles

Reverse Conformational Changes of the Light Chain-Binding Domain of Myosin V and VI Processive Motor Heads during and after Hydrolysis of ATP by Small-Angle X-Ray Solution Scattering

We used small-angle X-ray solution scattering (SAXS) technique to investigate the nucleotide-mediated conformational changes of the head domains [subfragment 1 (S1)] of myosin V and VI processive motors that govern their directional preference for motility on actin. Recombinant myosin V-S1 with two IQ motifs (MV-S1IQ2) and myosin VI-S1 (MVI-S1) were engineered from Sf9 cells using a baculovirus expression system. The radii of gyration ( R g) of nucleotide-free MV-S1IQ2 and MVI-S1 were 48.6 and 48.8 Å, respectively. In the presence of ATP, the R g value of MV-S1IQ2 decreased to 46.7 Å, while that of MVI-S1 increased to 51.7 Å, and the maximum chord length of the molecule decreased by ca 9% for MV-S1IQ2 and increased by ca 6% for MVI-S1. These opposite directional changes were consistent with those occurring in S1s with ADP and Vi or AlF 4 − 2 bound (i.e., in states mimicking the ADP/Pi-bound state of ATP hydrolysis). Binding of AMPPNP induced R g changes of both constructs similar to those in the presence of ATP, suggesting that the timing of the structural changes for their motion on actin is upon binding of ATP (the pre-hydrolysis state) during the ATPase cycle. Binding of ADP to MV-S1IQ2 and MVI-S1 caused their R g values to drop below those in the nucleotide-free state. Thus, upon the release of Pi, the reverse conformational change could occur, coupling to drive the directional motion on actin. The amount of R g change upon the release of Pi was ca 6.4 times greater in MVI-S1 than in MV-S1IQ2, relating to the production of the large stroke of the MVI motor during its translocation on actin. Atomic structural models for these S1s based upon the ab initio shape reconstruction from X-ray scattering data were constructed, showing that MVI-S1 has the light-chain-binding domain positioned in the opposite direction to MV-S1IQ2 in both the pre- and post-powerstroke transition. The angular change between the light chain-binding domains of MV-S1IQ2 in the pre- to post-powerstroke transition was ∼ 50°, comparable to that of MII-S1. On the other hand, that of MVI-S1 was ∼ 100° or ∼ 130° much less than the currently postulated changes to allow the maximal stroke size of myosin VI-S1 but still significantly larger than those of other myosins reported so far. The results suggest that some additional alterations or elements are required for MVI-S1 to take maximal working strokes along the actin filament.

Van't Hoff Analysis of ATP Binding to Human P-glycoprotein (ABCB1)

ATP binding to ABC transport proteins is an established step in the mechanism by which substrates are transported. Basal ATPase activity (activity seen in the absence of transported drugs) also facilitates multidrug transport by ABC proteins (Al-Shawi et al. 2003, J. Biol. Chem. 278, 52629). Therefore, we aimed to develop a non-perturbing method for detecting structural changes of P-glycoprotein associated with ATP binding during turnover. To accomplish this we analyzed heat capacity changes during ATP binding to P-glycoprotein by generating van't Hoff relationships for KMATP values for ATP binding and KiAMPPNP values for AMPPNP binding (non-hydrolysable analog of ATP).The rate of ATP hydrolysis by purified P-glycoprotein was measured as a function of ATP and AMPPNP concentrations at different temperatures from 23 to 35°C, in the presence or absence of saturating concentration of drug. Plots of Ln(KaATP) as a function of reciprocal absolute temperature in the presence of verapamil, valinomycin, colchicine and in absence of drug were fit by a nonlinear van't Hoff equation. Changes in heat capacity were derived from the fits. For basal activity, the Ln(KaATP) increased with reciprocal temperature. The heat capacity change was positive. In the presence of verapamil the relationship declined and the heat capacity change was negative. Results obtained with the other drugs were similar. This suggests that P-glycoprotein became more hydrated on ATP binding during basal activity and adopted a more open form. In contrast, in the presence of verapamil P-glycoprotein became less hydrated and adopted a more compact form. Overall, the data support our contention that there are two structurally distinct reaction pathways for ATP hydrolysis by ABC transporters and further help clarify the coupling mechanism of ATP hydrolysis to substrate transport. Supported by NIH grant GM52502.

Crystal Structures of the Lyn Protein Tyrosine Kinase Domain in Its Apo- and Inhibitor-bound State

The Src-family protein-tyrosine kinase (PTK) Lyn is the most important Src-family kinase in B cells, having both inhibitory and stimulatory activity that is dependent on the receptor, ligand, and developmental context of the B cell. An important role for Lyn has been reported in acute myeloid leukemia and chronic myeloid leukemia, as well as certain solid tumors. Although several Src-family inhibitors are available, the development of Lyn-specific inhibitors, or inhibitors with reduced off-target activity to Lyn, has been hampered by the lack of structural data on the Lyn kinase. Here we report the crystal structure of the non-liganded form of Lyn kinase domain, as well as in complex with three different inhibitors: the ATP analogue AMP-PNP; the pan Src kinase inhibitor PP2; and the BCR-Abl/Src-family inhibitor Dasatinib. The Lyn kinase domain was determined in its "active" conformation, but in the unphosphorylated state. All three inhibitors are bound at the ATP-binding site, with PP2 and Dasatinib extending into a hydrophobic pocket deep in the substrate cleft, thereby providing a basis for the Src-specific inhibition. Analysis of sequence and structural differences around the active site region of the Src-family PTKs were evident. Accordingly, our data provide valuable information for the further development of therapeutics targeting Lyn and the important Src-family of kinases.

Crystallographic Studies on Multiple Conformational States of Active-site Loops in Pyrrolysyl-tRNA Synthetase

Pyrrolysine, a lysine derivative with a bulky pyrroline ring, is the “22nd” genetically encoded amino acid. In the present study, the carboxy-terminal catalytic fragment of Methanosarcina mazei pyrrolysyl-tRNA synthetase (PylRS) was analyzed by X-ray crystallography and site-directed mutagenesis. The catalytic fragment ligated tRNA Pyl with pyrrolysine nearly as efficiently as the full-length PylRS. We determined the crystal structures of the PylRS catalytic fragment in the substrate-free, ATP analogue (AMPPNP)-bound, and AMPPNP/pyrrolysine-bound forms, and compared them with the previously-reported PylRS structures. The ordering loop and the motif-2 loop undergo conformational changes from the “open” states to the “closed” states upon AMPPNP binding. On the other hand, the β7-β8 hairpin exhibits multiple conformational states, the open, intermediate (β7-open/β8-open and β7-closed/β8-open), and closed states, which are not induced upon substrate binding. The PylRS structures with a docked tRNA suggest that the active-site pocket can accommodate the CCA terminus of tRNA when the motif-2 loop is in the closed state and the β7-β8 hairpin is in the open or intermediate state. The entrance of the active-site pocket is nearly closed in the closed state of the β7-β8 hairpin, which may protect the pyrrolysyladenylate intermediate in the absence of tRNA Pyl. Moreover, a structure-based mutational analysis revealed that hydrophobic residues in the amino acid-binding tunnel are important for accommodating the pyrrolysine side chain and that Asn346 is essential for anchoring the side-chain carbonyl and α-amino groups of pyrrolysine. In addition, a docking model of PylRS with tRNA was constructed based on the aspartyl-tRNA synthetase/tRNA structure, and was confirmed by a mutational analysis.

Thermodynamics of the ATPase Cycle of GlcV, the Nucleotide-Binding Domain of the Glucose ABC Transporter of Sulfolobus solfataricus

ATP-binding cassette transporters drive the transport of substrates across the membrane by the hydrolysis of ATP. They typically have a conserved domain structure with two membrane-spanning domains that form the transport channel and two cytosolic nucleotide-binding domains (NBDs) that energize the transport reaction. Binding of ATP to the NBD monomer results in formation of a NBD dimer. Hydrolysis of the ATP drives the dissociation of the dimer. The thermodynamics of distinct steps in the ATPase cycle of GlcV, the NBD of the glucose ABC transporter of the extreme thermoacidophile Sulfolobus solfataricus, were studied by isothermal titration calorimetry using the wild-type protein and two mutants, which are arrested at different steps in the ATP hydrolytic cycle. The G144A mutant is unable to dimerize, while the E166A mutant is defective in dimer dissociation. The ATP, ADP, and AMP-PNP binding affinities, stoichiometries, and enthalpies of binding were determined at different temperatures. From these data, the thermodynamic parameters of nucleotide binding, NBD dimerization, and ATP hydrolysis were calculated. The data demonstrate that the ATP hydrolysis cycle of isolated NBDs consists of consecutive steps where only the final step of ADP release is energetically unfavorable.

Functional Characterization of Two Nucleotide-binding Sites in Soluble Guanylate Cyclase

Soluble guanylate cyclase is a heterodimeric hemoprotein composed of alpha- and beta-subunits with a homologous motif to the nucleotide-binding sites of adenylate cyclases. Homology modeling of guanylate cyclase, based on the crystal structure of adenylate cyclase, reveals a single GTP-binding site and a putative second site pseudosymmetric to the GTP-binding site. However, the role of this pseudosymmetric site has remained unclear. Using equilibrium dialysis, we identified two nucleotide-binding sites with high and low affinity for alpha,beta-methylene guanosine 5'-triphosphate (GMP-CPP). In contrast, 2'-dADP occupied both sites with equivalent affinities. Adenosine-5'-beta,gamma-imido triphosphate (AMP-PNP), which competitively inhibited the cyclase reaction, bound solely to the high affinity site, indicating the role of this site as the catalytic site. The function of the low affinity site was examined using allosteric activators YC-1 and BAY 41-2272. YC-1 significantly reduced the affinity of 2'-dADP, probably by competing for the same site as 2'-dADP. BAY 41-2272 totally inhibited the specific binding of one molecule of 2'-dADP as well as GMP-CPP. This suggests that the activators compete with these nucleotides for the low affinity site. Infrared and EPR analyses of the enzymic CO- and NO-hemes also supported the suggested role of the low affinity site as a target for the activators. Our results imply that the low affinity site is the pseudosymmetric site, which binds YC-1 or BAY 41-2272.

Hydrogen Exchange Solvent Protection by an ATP Analogue Reveals Conformational Changes in ERK2 upon Activation

Structural and kinetic studies have provided extensive information about the molecular mechanisms of kinase activation by phosphorylation. However, it is still unclear how changes in protein dynamics and flexibility contribute to catalytic function. Mass spectrometry was used to probe changes in hydrogen/deuterium exchange in the MAP kinase, ERK2, in the presence and absence of the ATP analogue, AMP-PNP. In both active and inactive forms of ERK2, protection from hydrogen exchange by AMP-PNP binding was observed within conserved ATP binding motifs in the N-terminal lobe, which are known to directly interact with nucleotide in various protein kinases. In contrast, higher protection from exchange by AMP-PNP was observed in active ERK2 compared to inactive ERK2, in a region corresponding to the conserved DFG motif, which is located in the C-terminal lobe and coordinates Mg2+ at the catalytic site. Thus, AMP-PNP binding simultaneously protects residues within the N and C terminus in the active form of ERK2, but not the inactive form. This demonstrates that ERK2 binds nucleotide in two modes, in which active ERK2 adopts a closed conformation following nucleotide binding in solution, while inactive ERK2 adopts an open conformation. The finding provides novel evidence that phosphorylation of ERK2 facilitates interdomain closure, allowing proper orientation between ATP and substrate to facilitate phosphoryl transfer.

Probing noncovalent protein–ligand interactions of the cgmp-dependent protein kinase using electrospray ionization time of flight mass spectrometry

Nanoflow electrospray ionization time of flight mass spectrometry (ESI-TOF-MS) was used to study activation properties of the cGMP-dependent protein kinase (PKG). Our nanoflow ESI-TOF-MS analysis confirms that PKG mainly occurs as a 153 kDa homodimer and is able to bind four cGMP molecules, which is in agreement with the known stoichiometry. Binding order and stoichiometry of cGMP, the non-hydrolysable ATP analog beta,gamma-imidoadenosine 5'-triphosphate (AMPPNP) and Mn2+ for PKG were characterized as model for the active PKG-cGMP-ATP/Mg2+ complex. Already in the absence of cGMP, a noncovalent complex between PKG and two molecules of AMPPNP could be observed by ESI-TOF-MS. Binding of AMPPNP to PKG was strongly enhanced by the addition of MnCl2 to the spray solution. This is in agreement with binding of AMPPNP/Mn2+ in the ATP binding pocket of PKG since all protein kinases require a metal ion to accompany ATP in the ATP-binding pocket for proper positioning of the beta and gamma phosphates. Additionally, this finding could imply that within the inactive conformation of PKG, the autoinhibition-domain, when in contact with the substrate-docking domain, does not block the entrance to the ATP-binding site. In the presence of cGMP, less of the fully saturated PKG-(cGMP)4(AMPPNP/Mn2+)2 complex was observed, suggesting that the PKG-ATP interaction is weakened in the active conformation of PKG. Additionally, limited proteolysis in combination with native-ESI MS showed to be a useful tool to study the contact regions on the PKG-dimer and also allowed the rapid determination of the overall autophosphorylation status of the protein. These measurements indicated that autophosphorylation mainly occurs within the first 80 aminoterminal residues and involves in total 3-4 phosphates per subunit.

Inter-domain Motions of the N-domain of the KdpFABC Complex, a P-type ATPase, are not Driven by ATP-induced Conformational Changes

P-type ATPases are involved in the active transport of ions across biological membranes. The KdpFABC complex (P-type ATPase) of Escherichia coli is a high-affinity K+ uptake system that operates only when the cell experiences osmotic stress or K+ limitation. Here, we present the solution structure of the nucleotide binding domain of KdpB (backbone RMSD 0.17 A) and a model of the AMP-PNP binding mode based on intermolecular distance restraints. The calculated AMP-PNP binding mode shows the purine ring of the nucleotide to be "clipped" into the binding pocket via a pi-pi-interaction to F377 on one side and a cation-pi-interaction to K395 on the other. This binding mechanism seems to be conserved in all P-type ATPases, except the heavy metal transporting ATPases (type IB). Thus, we conclude that the Kdp-ATPase (currently type IA) is misgrouped and has more similarities to type III ATPases. The KdpB N-domain is the smallest and simplest known for a P-type ATPase, and represents a minimal example of this functional unit. No evidence of significant conformational changes was observed within the N-domain upon nucleotide binding, thus ruling out a role for ATP-induced conformational changes in the reaction cycle.

Bioinformatics analysis of two-component regulatory systems in Staphylococcus epidermidis

Sixteen pairs of two-component regulatory systems are identified in the genome of Staphylococcus epidermidis ATCC12228 strain, which is newly sequenced by our laboratory for Medical Molecular Virology and Chinese National Human Genome Center at Shanghai, by using bioinformatics analysis. Comparative analysis of the twocomponent regulatory systems in S. epidermidis and that of S. aureus and Bacillus subtilis shows that these systems may regulate some important biological functions, e.g. growth, biofilm formation, and expression of virulence factors in S. epidermidis. Two conserved domains, i.e. HATPase c and REC domains, are found in all 16 pairs of two-component proteins. Homologous modelling analysis indicates that there are 4 similar HATPase c domain structures of histidine kinases and 13 similar REC domain structures of response regulators, and there is one AMP-PNP binding pocket in the HATPase c domain and three active aspartate residues in the REC domain. Preliminary experiment reveals that the bioinformatics analysis of the conserved domain structures in the two-component regulatory systems in S. epidermidis may provide useful information for discovery of potential drug target.

TNP-AMP Binding to the Sarcoplasmic Reticulum Ca 2+-ATPase Studied by Infrared Spectroscopy

Infrared spectroscopy was used to monitor the conformational change of 2′,3′- O-(2,4,6-trinitrophenyl)adenosine 5′-monophosphate (TNP-AMP) binding to the sarcoplasmic reticulum Ca 2+-ATPase. TNP-AMP binding was observed in a competition experiment: TNP-AMP is initially bound to the ATPase but is then replaced by β, γ-iminoadenosine 5′-triphosphate (AMPPNP) after AMPPNP release from P 3-1-(2-nitrophenyl)ethyl AMPPNP (caged AMPPNP). The resulting infrared difference spectra are compared to those of AMPPNP binding to the free ATPase, to obtain a difference spectrum that reflects solely TNP-AMP binding to the Ca 2+-ATPase. TNP-AMP used as an ATP analog in the crystal structure of the sarcoplasmic reticulum Ca 2+-ATPase was found to induce a conformational change upon binding to the ATPase. It binds with a binding mode that is different from that of AMPPNP, ATP, and other tri- and diphosphate nucleotides: TNP-AMP binding causes partially opposite and smaller conformational changes compared to ATP or AMPPNP. The conformation of the TNP-AMP ATPase complex is more similar to that of the E1Ca 2 state than to that of the E1ATPCa 2 state. Regarding the use of infrared spectroscopy as a technique for ligand binding studies, our results show that infrared spectroscopy is able to distinguish different binding modes.

NMR chemical shift perturbation study of the N-terminal domain of Hsp90 upon binding of ADP, AMP-PNP, geldanamycin, and radicicol.

Hsp90 is one of the most abundant chaperone proteins in the cytosol. In an ATP-dependent manner it plays an essential role in the folding and activation of a range of client proteins involved in signal transduction and cell cycle regulation. We used NMR shift perturbation experiments to obtain information on the structural implications of the binding of AMP-PNP (adenylyl-imidodiphosphate-a non-hydrolysable ATP analogue), ADP and the inhibitors radicicol and geldanamycin. Analysis of (1)H,(15)N correlation spectra showed a specific pattern of chemical shift perturbations at N210 (ATP binding domain of Hsp90, residues 1-210) upon ligand binding. This can be interpreted qualitatively either as a consequence of direct ligand interactions or of ligand-induced conformational changes within the protein. All ligands show specific interactions in the binding site, which is known from the crystal structure of the N-terminal domain of Hsp90. For AMP-PNP and ADP, additional shift perturbations of residues outside the binding pocket were observed and can be regarded as a result of conformational rearrangement upon binding. According to the crystal structures, these regions are the first alpha-helix and the "ATP-lid" ranging from amino acids 85 to 110. The N-terminal domain is therefore not a passive nucleotide-binding site, as suggested by X-ray crystallography, but responds to the binding of ATP in a dynamic way with specific structural changes required for the progression of the ATPase cycle.

The Role of the Invariant His-1069 in Folding and Function of the Wilson's Disease Protein, the Human Copper-transporting ATPase ATP7B

The copper-transporting ATPase ATP7B is essential for normal distribution of copper in human cells. Mutations in ATP7B lead to Wilson's disease, a severe disorder with neurological and hepatic manifestations. One of the most common disease mutations, a H1069Q substitution, causes intracellular mislocalization of ATP7B (the Wilson's disease protein, WNDP). His-1069 is located in the nucleotide-binding domain of WNDP and is conserved in all copper-transporting ATPases from bacteria to mammals; however, the specific role of this His in the structure and function of WNDP remains unclear. We demonstrate that substitution of His-1069 for Gln, Ala, or Cys does not significantly alter the folding of the WNDP nucleotide-binding domain or the proteolytic resistance of the full-length WNDP. In contrast, the function of WNDP is markedly affected by the mutations. The ability to form an acylphosphate intermediate in the presence of ATP is entirely lost in all three mutants, suggesting that His-1069 is important for ATP-dependent phosphorylation. Other steps of the WNDP enzymatic cycle are less dependent on His-1069. The H1069C mutant shows normal phosphorylation in the presence of inorganic phosphate; it binds an ATP analogue, beta,gamma-imidoadenosine 5'-triphosphate (AMP-PNP), and copper and undergoes nucleotide-dependent conformational transitions similar to those of the wild-type WNDP. Although binding of AMP-PNP is not disrupted by the mutation, the apparent affinity for the nucleotide is decreased by 4-fold. We conclude that His-1069 is responsible for proper orientation of ATP in the catalytic site of WNDP prior to ATP hydrolysis.

The RNA Helicase DbpA Exhibits a Markedly Different Conformation in the ADP-bound State When Compared with the ATP- or RNA-Bound States

The motor enzymes that belong to the family of RNA helicases catalyze the strand separation of duplex RNA via ATP hydrolysis. Among these enzymes, Escherichia coli DbpA is a unique RNA helicase because it possesses ATPase-specific activity toward the peptidyl transferase center in 23 S ribosomal RNA. For this reason, it has been the subject of numerous biochemical and structure-function studies. The ATP-stimulated unwinding activity of DbpA toward specific and nonspecific RNA duplexes has been demonstrated. However, the underlying molecular and structural basis, which facilitates its helicase activities, is presently not known. We combined time-dependent limited proteolysis digestion, fluorescence spectroscopy, and three-dimensional structural homology modeling techniques to study the structural conformations of DbpA with respect to its binding to stoichiometric ratios of RNA and cofactors. We show that the conformational state of DbpA is markedly different in the ADP-bound state than in any other state (ATP- or RNA-bound). These results, together with structural homology studies, suggest that a hinge region located in the core domain of DbpA mediates such conformational changes.

ATP analogue binding to the A subunit induces conformational changes in the E subunit that involves a disulfide bond formation in plant V-ATPase.

Vacuolar H+-ATPase (V-ATPase) consists of a catalytic head, a stalk part and a membrane domain. We indirectly investigated the interaction between the A subunit (catalytic head) and the E subunit (stalk part) using an ATP analogue, adenosine 5'-[beta,gamma-imino]triphosphate (AMP-PNP), which holds the enzyme in the substrate-binding state. AMP-PNP treatment caused a mobility shift of the E subunit with a faster migration in SDS/polyacrylamide gel electrophoresis without a reductant, while ATP treatment did not. A mobility shift of the E subunit has been detected in several plants. As polypeptides with intramolecular disulfide bonds migrate faster than those without disulfide bonds, the mobility shift may be due to the formation of an intramolecular disulfide bond by two cysteine residues conserved among several plant species. The mobility shift may be involved in the binding of AMP-PNP to the ATP-binding site, which exists in the A and B subunits, as it was inhibited by the addition of ATP. Pretreatment with 2'-3'-O-(4-benzoylbenzoyl)-ATP (Bz-ATP), which modifies the ATP-binding site of the B subunit under UV illumination, did not inhibit the mobility shift of the E subunit caused by AMP-PNP treatment. The response of V-ATPase following the AMP-PNP binding may cause a conformational change in the E subunit into a form that is susceptible to oxidation of cysteine residues. This is the first demonstration of interaction between the A and E subunits in the substrate-binding state of a plant V-ATPase.

Severed channels probe regulation of gating of cystic fibrosis transmembrane conductance regulator by its cytoplasmic domains.

Opening and closing of a CFTR Cl(-) channel is controlled by PKA-mediated phosphorylation of its cytoplasmic regulatory (R) domain and by ATP binding, and likely hydrolysis, at its two nucleotide binding domains. Functional interactions between the R domain and the two nucleotide binding domains were probed by characterizing the gating of severed CFTR channels expressed in Xenopus oocytes. Expression levels were assessed using measurements of oocyte conductance, and detailed functional characteristics of the channels were extracted from kinetic analyses of macroscopic current relaxations and of single-channel gating events in membrane patches excised from the oocytes. The kinetic behavior of wild-type (WT) CFTR channels was compared with that of split CFTR channels bearing a single cut (between residues 633 and 634) just before the R domain, of split channels with a single cut (between residues 835 and 837) just after the R domain, and of split channels from which the entire R domain (residues 634-836) between those two cut sites was omitted. The channels cut before the R domain had characteristics almost identical to those of WT channels, except for less than twofold shorter open burst durations in the presence of PKA. Channels cut just after the R domain were characterized by a low level of activity even without phosphorylation, strong stimulation by PKA, enhanced apparent affinity for ATP as assayed by open probability, and a somewhat destabilized binding site for the locking action of the nonhydrolyzable ATP analog AMPPNP. Split channels with no R domain (from coexpression of CFTR segments 1-633 and 837-1480) were highly active without phosphorylation, but otherwise displayed the characteristics of channels cut after the R domain, including higher apparent ATP affinity, and less tight binding of AMPPNP at the locking site, than for WT. Intriguingly, severed channels with no R domain were still noticeably stimulated by PKA, implying that activation of WT CFTR by PKA likely also includes some component unrelated to the R domain. As the maximal opening rates were the same for WT channels and split channels with no R domain, it seems that the phosphorylated R domain does not stimulate opening of CFTR channels; rather, the dephosphorylated R domain inhibits them.

Conformational Changes of H+ ATP Synthase Upon ATP Hydrolysis Detected by Fluorescence Correlation Spectroscopy

H+ -ATPases catalyze the synthesis of ATP from ADP and phosphate in the membranes of mitochondria, chloroplasts and bacteria. The enzyme consists of two parts: the hydrophobic, membrane integrated F0-part is involved in proton transport and contains the subunits a, b and c with a likely stoichiometry ab2c12 for the Escherichia coli enzyme. The nucleotide binding sites are located in the hydrophilic F1-part with subunit composition α3β3γδε [1]. During ATP hydrolysis intersubunit rotation of the γ-subunit in the F1-part (which was dissociated from the holoenzyme) was observed with single fluorescence labeled enzymes in realtime using enhanced videomicroscopy [2 - 4]. ATP synthesis and hydrolysis occur at the catalytic binding sites of the β-subunits. In the crystal structure of the F1-ATPase three different conformations of the three β-subunits were detected [5]. From these structural data a detailed mechanistic model of the F1-ATPase as a ‘stepped rotatory motor protein’ was developed [6]. According to this model, large conformational changes in the lower part of the β-subunits are expected during catalysis. We investigated the diffusion of the F1-part before and after binding of ATP with fluorescence correlation spectroscopy (FCS). The β-subunit was specifically labeled with a newly synthesized sulfonyl fluoride derivative of Sulforhodamine G. The labeled amino acid is not known yet. Preliminary FCS measurements with EF1 are shown (Fig.1.). Upon ATP binding the translational diffusion time is decreased by about 15 percent due to changes in size and shape of the enzymes during the catalytic cycle. Similar results were obtained for EF1-ATPase with a fluorescence label on the γ-subunit [7]. These FCS measurements are supported by new electron microscopy data, which show a significant shrinking up of F1-part of the enzyme upon binding of the non-hydrolysable ATP derivative AMPPNP. The diameter of the F1-part decreased mainly in the upper half of the F1-part upon binding of AMPPNP [8].

Direct visualisation of conformational changes in EF0F1 by electron microscopy

The isolated H+-ATPase from Escherichia coli (EF0F1) was investigated by electron microscopy of samples of negatively stained monodisperse molecules, followed by single-particle image processing. The resulting three-dimensional maps showed that the F1-part is connected by a prominent stalk to a more peripheral part of F0. The F1-part showed stain-accessible cavities inside. In three-dimensional maps from selected particles, a second stalk could be detected which was thinner than the main stalk and is thought to correspond to the stator.Three-dimensional maps of the enzyme in the absence and in the presence of the substrate analogue adenyl-β,γ-imidodiphosphate (AMP-PNP) were calculated. Upon binding of AMP-PNP the three-dimensional maps showed no significant changes in the F0-part of EF0F1, whereas a major conformational change in the F1-part was observed. (1) The diameter of the F1-part decreased upon binding of AMP-PNP mainly in the upper half of F1. (2) Enzyme particles prepared in the presence of AMP-PNP had a pointed cap at the top of the F1-part which was missing in its absence. (3) The stain-accessible cavity inside the F1-part altered its pattern significantly.

Binding of non-catalytic ATP to human hexokinase I highlights the structural components for enzyme-membrane association control.

Hexokinase I sets the pace of glycolysis in the brain, catalyzing the ATP-dependent phosphorylation of glucose. The catalytic properties of hexokinase I are dependent on product inhibition as well as on the action of phosphate. In vivo, a large fraction of hexokinase I is bound to the mitochondrial outer membrane, where the enzyme adopts a tetrameric assembly. The mitochondrion-bound hexokinase I is believed to optimize the ATP/ADP exchange between glucose phosphorylation and the mitochondrial oxidative phosphorylation reactions. The crystal structure of human hexokinase I has been determined at 2.25 A resolution. The overall structure of the enzyme is in keeping with the closed conformation previously observed in yeast hexokinase. One molecule of the ATP analogue AMP-PNP is bound to each N-terminal domain of the dimeric enzyme in a surface cleft, showing specific interactions with the nucleotide, and localized positive electrostatic potential. The molecular symmetry brings the two bound AMP-PNP molecules, at the centre of two extended surface regions, to a common side of the dimeric hexokinase I molecule. The binding of AMP-PNP to a protein site separated from the catalytic centre of human hexokinase I can be related to the role played by some nucleotides in dissociating the enzyme from the mitochondrial membrane, and helps in defining the molecular regions of hexokinase I that are expected to be in contact with the mitochondrion. The structural information presented here is in keeping with monoclonal antibody mapping of the free and mitochondrion-bound forms of the enzyme, and with sequence analysis of hexokinases that differ in their mitochondria binding properties.

A pre-steady-state kinetic analysis of substrate binding to human recombinant deoxycytidine kinase: a model for nucleoside kinase action.

Deoxycytidine kinase (dCK) is an enzyme with broad substrate specificity which can phosphorylate pyrimidine and purine deoxynucleosides, including important antiviral and cytostatic agents. In this study, stopped-flow experiments were used to monitor intrinsic fluorescence changes induced upon binding of various phosphate donors (ATP, UTP, and the nonhydrolyzable analogue AMP-PNP) and the acceptor dCyd to recombinant dCK. Monophasic kinetics were observed throughout. The nucleotides as well as dCyd bound to the enzyme by a two-step mechanism, involving a rapid initial equilibrium step, followed by a protein conformational change that is responsible for the fluorescence change. The bimolecular association rate constants for nucleotide binding [(4-10) x 10(3) M-1 s-1] were 2-3 orders of magnitude lower than those for dCyd binding [(1.3-1.5 x 10(6) M-1 s-1]. This difference most likely is due predominantly to the large difference in the forward rate constants of the conformational changes (0.04-0.26 s-1 vs 560-710 s-1). Whereas the kinetics of the binding of ATP, UTP, and AMP-PNP to dCK showed some differences, UTP exhibiting the tightest binding, no significant differences were observed for the binding of dCyd to dCK in the presence or absence of phosphate donors. However, the binding of dCyd to dCK in the presence of ATP or UTP was accompanied by a 1.5- or 3-fold higher quenching amplitude as compared with dCyd alone or in the presence of AMP-PNP. We conclude that ATP and UTP induce a conformational change in the enzyme, thereby enabling efficient phosphoryl transfer.