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Pyruvate dehydrogenase kinase (PDK) controls the activity of pyruvate decarboxylase complex (PDC) by phosphorylating key serine residues on the E1 subunit, which leads to a decreased oxidative phosphorylation in mitochondria. Inhibition of PDK activity by natural/synthetic compounds has been shown to reverse the Warburg effect, a characteristic metabolism in cancer cells. PDK-PDC axis also has been associated with diabetes and heart disease. Therefore, regulation of PDK activity has been considered as a promising strategy to treat related diseases. Here we present the X-ray crystal structure of PDK2 complexed with a recently identified PDK4 inhibitor, compound 8c, which has been predicted to bind at the lipoyl-binding site and interrupt intermolecular interactions with the E2-E3bp subunits of PDC. The co-crystal structure confirmed the specific binding location of compound 8c and revealed the remote conformational change in the ATP-binding pocket. In addition, two novel 4,5-diarylisoxazole derivatives, GM10030 and GM67520, were synthesized and used for structural studies, which target the ATP-binding site of PDK2. These compounds bind to PDK2 with a sub-100nM affinity as determined by isothermal titration calorimetry experiments. Notably, the crystal structure of the PDK2-GM10030 complex displays unprecedented asymmetric conformation of human PDK2 dimer, especially in the ATP-lids and C-terminal tails., (Copyright © 2020 Elsevier Inc. All rights reserved.)

The response of ATP-sensitive K + channels (K ATP ) to cellular metabolism is coordinated by three classes of nucleotide binding site (NBS). We used a novel approach involving labeling of intact channels in a native, membrane environment with a non-canonical fluorescent amino acid and measurement (using FRET with fluorescent nucleotides) of steady-state and time-resolved nucleotide binding to dissect the role of NBS2 of the accessory SUR1 subunit of K ATP in channel gating. Binding to NBS2 was Mg 2+ -independent, but Mg 2+ was required to trigger a conformational change in SUR1. Mutation of a lysine (K1384A) in NBS2 that coordinates bound nucleotides increased the EC 50 for trinitrophenyl-ADP binding to NBS2, but only in the presence of Mg 2+ , indicating that this mutation disrupts the ligand-induced conformational change. Comparison of nucleotide-binding with ionic currents suggests a model in which each nucleotide binding event to NBS2 of SUR1 is independent and promotes K ATP activation by the same amount., Competing Interests: MP, NV, SU, FA No competing interests declared, (© 2019, Puljung et al.)

MLKL is a pore forming pseudokinase involved in the final stage of necroptosis, a form of programmed cell death. Its phosphorylation by RIPK3 is necessary for triggering necroptosis but not for triggering apoptosis, which makes it a unique target for pharmacological inhibition to block necroptotic cell death. This mechanism has been described as playing a role in disease progression in neurodegenerative and inflammatory diseases. A type II kinase inhibitor (cpd 1) has been described that reportedly binds to the MLKL pseudokinase domain and prevents necroptosis. Here we describe five compounds that bind to the MLKL ATP-binding site, however the four MLKL-selective compounds have no activity in rescuing cells from necroptosis. We use kinase selectivity panels, crystallography and a new conformationally sensitive method of measuring protein conformational changes (SHG) to confirm that the one previously reported compound that can rescue cells (cpd 1) is a non-selective type II inhibitor that also inhibits the upstream kinase RIPK1. Although this compound can shift the GFE motif of the activation loop to an "out" position, the accessibility of the key residue Ser358 in the MLKL activation loop is not affected by compound binding to the MLKL active site. Our studies indicate that an ATP-pocket inhibitor of the MLKL pseudokinase domain does not have any impact on the necroptosis pathway, which is contrary to a previously reported study., Competing Interests: We declare that the authors of this manuscript are employed by Biogen or Biodesy (as described in the author list) and have no patents in development with respect to this work. Our commercial affiliation does not alter our adherence to all PLOS ONE policies on sharing data and materials.

The P-glycoprotein (Pgp) transporter plays a central role in drug disposition by effluxing a chemically diverse range of drugs from cells through conformational changes and ATP hydrolysis. A number of drugs are known to activate ATP hydrolysis of Pgp, but coupling between ATP and drug binding is not well understood. The cardiovascular drug verapamil is one of the most widely studied Pgp substrates and therefore, represents an ideal drug to investigate the drug-induced ATPase activation of Pgp. As previously noted, verapamil-induced Pgp-mediated ATP hydrolysis kinetics was biphasic at saturating ATP concentrations. However, at subsaturating ATP concentrations, verapamil-induced ATPase activation kinetics became monophasic. To further understand this switch in kinetic behavior, the Pgp-coupled ATPase activity kinetics was checked with a panel of verapamil and ATP concentrations and fit with the substrate inhibition equation and the kinetic fitting software COPASI. The fits suggested that cooperativity between ATP and verapamil switched between low and high verapamil concentration. Fluorescence spectroscopy of Pgp revealed that cooperativity between verapamil and a non-hydrolyzable ATP analog leads to distinct global conformational changes of Pgp. NMR of Pgp reconstituted in liposomes showed that cooperativity between verapamil and the non-hydrolyzable ATP analog modulate each other's interactions. This information was used to produce a conformationally-gated model of drug-induced activation of Pgp-mediated ATP hydrolysis., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Aminoacyl-tRNA synthetases recognize cognate amino acids and tRNAs from their noncognate counterparts and catalyze the formation of aminoacyl-tRNAs. Halofuginone (HF), a coccidiostat used in veterinary medicine, exerts its effects by acting as a high-affinity inhibitor of the enzyme glutamyl-prolyl-tRNA synthetase (EPRS). In order to elucidate the precise molecular basis of this inhibition mechanism of human EPRS, the crystal structures of the prolyl-tRNA synthetase domain of human EPRS (hPRS) at 2.4 Å resolution (hPRS-apo), of hPRS complexed with ATP and the substrate proline at 2.3 Å resolution (hPRS-sub) and of hPRS complexed with HF at 2.62 Å resolution (hPRS-HF) are presented. These structures show plainly that motif 1 functions as a cap in hPRS, which is loosely opened in hPRS-apo, tightly closed in hPRS-sub and incorrectly closed in hPRS-HF. In addition, the structural analyses are consistent with more effective binding of hPRS to HF with ATP. Mutagenesis and biochemical analysis confirmed the key roles of two residues, Phe1097 and Arg1152, in the HF inhibition mechanism. These structures will lead to the development of more potent and selective hPRS inhibitors for promoting inflammatory resolution.

Group II chaperonins play important roles in protein homeostasis in the eukaryotic cytosol and in Archaea. These proteins assist in the folding of nascent polypeptides and also refold unfolded proteins in an ATP-dependent manner. Chaperonin-mediated protein folding is dependent on the closure and opening of a built-in lid, which is controlled by the ATP hydrolysis cycle. Recent structural studies suggest that the ring structure of the chaperonin twists to seal off the central cavity. In this study, we demonstrate ATP-dependent dynamics of a group II chaperonin at the single-molecule level with highly accurate rotational axes views by diffracted X-ray tracking (DXT). A UV light-triggered DXT study with caged-ATP and stopped-flow fluorometry revealed that the lid partially closed within 1 s of ATP binding, the closed ring subsequently twisted counterclockwise within 2-6 s, as viewed from the top to bottom of the chaperonin, and the twisted ring reverted to the original open-state with a clockwise motion. Our analyses clearly demonstrate that the biphasic lid-closure process occurs with unsynchronized closure and a synchronized counterclockwise twisting motion.

The cystic fibrosis transmembrane conductance regulator (CFTR) acts as a channel on the apical membrane of epithelia. Disease-causing mutations in the cystic fibrosis gene can lead to CFTR protein misfolding as in the case of the F508del mutation and/or channel dysfunction. Recently, a small molecule, VX-770 (ivacaftor), has shown efficacy in restoring lung function in patients bearing the G551D mutation, and this has been linked to repair of its channel gating defect. However, these studies did not reveal the mechanism of action of VX-770 in detail. Normally, CFTR channel activity is regulated by phosphorylation, ATP binding, and hydrolysis. Hence, it has been hypothesized that VX-770 modifies one or more of these metabolic events. In this study, we examined VX-770 activity using a reconstitution system for purified CFTR protein, a system that enables control of known regulatory factors. We studied the consequences of VX-770 interaction with CFTR incorporated in planar lipid bilayers and in proteoliposomes, using a novel flux-based assay. We found that purified and phosphorylated CFTR was potentiated in the presence of Mg-ATP, suggesting that VX-770 bound directly to the CFTR protein, rather than associated kinases or phosphatases. Interestingly, we also found that VX-770 enhanced the channel activity of purified and mutant CFTR in the nominal absence of Mg-ATP. These findings suggest that VX-770 can cause CFTR channel opening through a nonconventional ATP-independent mechanism. This work sets the stage for future studies of the structural properties that mediate CFTR gating using VX-770 as a probe.

The protein kinase field is a very active research area in the pharmaceutical industry and many activities are ongoing to identify inhibitors of these proteins. The design of new chemical entities with improved pharmacological properties requires a deeper understanding of the factors that modulate inhibitor-kinase interactions. In this report, we studied the effect of two of these factors--the magnesium ion cofactor and the protein substrate--on inhibitors of the type I insulin-like growth factor receptor. Our results show that the concentration of magnesium ion influences the potency of adenosine triphosphate (ATP) competitive inhibitors, suggesting an explanation for the observation that such compounds retain their nanomolar potency in cells despite the presence of millimolar levels of ATP. We also showed that the peptidic substrate affects the potency of these inhibitors in a different manner, suggesting that the influence of this substrate on compound potency should be taken into consideration during drug discovery.

Glucokinase (GK) is the central player in glucose-stimulated insulin release from pancreatic β-cells, and catalytic activation by α-D-glucose binding has a key regulatory function. Whereas the mechanism of this activation is well understood, on the basis of crystal structures of human GK, there are no similar structural data on ATP binding to the ligand-free enzyme and how it affects its conformation. Here, we report on a conformational change induced by the binding of adenine nucleotides to human pancreatic GK, as determined by intrinsic tryptophan fluorescence, using the catalytically inactive mutant form T228M to correct for the inner filter effect. Adenosine-5'-(β,γ-imido)triphosphate and ATP bind to the wild-type enzyme with apparent [L](0.5) (ligand concentration at half-maximal effect) values of 0.27±0.02 mm and 0.78±0.14 mm, respectively. The change in protein conformation was further supported by ATP inhibition of the binding of the fluorescent probe 8-anilino-1-naphthalenesulfonate and limited proteolysis by trypsin, and by molecular dynamic simulations. The simulations provide a first insight into the dynamics of the binary complex with ATP, including motion of the flexible surface/active site loop and partial closure of the active site cleft. In the complex, the adenosine moiety is packed between two α-helices and stabilized by hydrogen bonds (with Thr228, Thr332, and Ser336) and hydrophobic interactions (with Val412 and Leu415). Combined with enzyme kinetic analyses, our data indicate that the ATP-induced changes in protein conformation may have implications for the kinetic cooperativity of the enzyme., (© 2011 The Authors Journal compilation © 2011 FEBS.)

Because mutations in RAS and BRAF represent the most common mutations found in human tumors, identification of inhibitors has been a major goal. Surprisingly, new oncogenic BRAF specific inhibitors inhibit cells transformed with mutated BRAF but paradoxically stimulate the growth of cells transformed with RAS. Here, we show that the mechanism for activation is via drug-induced dimer formation between CRAF and kinase suppressor of Ras (KSR)1. To understand the function of KSR1, we generated a KSR1 mutant that cannot bind ATP but stabilizes the closed, active conformation of KSR1. Molecular modeling suggested that the mutant stabilizes the two hydrophobic spines critical for the closed active conformation. We, therefore, could use the mutant to discriminate between the scaffold versus kinase functions of KSR1. The KSR1 mutant bound constitutively to RAF and mitogen-activated protein kinase kinase (MEK) but could not reconstitute activity suggesting that the catalytic activity of KSR1 is required for its function. Analogous mutations in BRAF and CRAF allowed us to test the generality of the model. The mutation induced changes consistent with the active, closed conformation of both kinases and confirmed that BRAF functions distinctly from CRAF in the MAP kinase pathway. Not only does this work suggest that KSR1 may function as a kinase, we anticipate that the mutation that we generated may be broadly applicable to stabilize the closed conformation of other kinases many of which may also form dimers.

Abl kinase inhibitors targeting the ATP binding pocket are currently employed as potent anti-leukemogenic agents but drug resistance has become a significant clinical limitation. Recently, a compound that binds to the myristate pocket of Abl (GNF-5) was shown to act cooperatively with nilotinib, an ATP-competitive inhibitor to target the recalcitrant "T315I" gatekeeper mutant of Bcr-Abl. To uncover an explanation for how drug binding at a distance from the kinase active site could lead to inhibition and how inhibitors could combine their effects, hydrogen exchange mass spectrometry (HX MS) was employed to monitor conformational effects in the presence of both dasatinib, a clinically approved ATP-site inhibitor, and GNF-5. While dasatinib binding to wild type Abl clearly influenced Abl conformation, no binding was detected between dasatinib and T315I. GNF-5, however, elicited the same conformational changes in both wild type and T315I, including changes to dynamics within the ATP site located approximately 25 Å from the site of GNF-5 interaction. Simultaneous binding of dasatinib and GNF-5 to T315I caused conformational and/or dynamics changes in Abl such that effects of dasatinib on T315I were the same as when it bound to wild type Abl. These results provide strong biophysical evidence that allosteric interactions play a role in Abl kinase downregulation and that targeting sites outside the ATP binding site can provide an important pharmacological tool to overcome mutations that cause resistance to ATP-competitive inhibitors.

Previous work conducted in our laboratories established the notion that TPGS 1000 (d-alpha-tocopheryl polyethylene glycol 1000 succinate), a nonionic surfactant, modulates P-glycoprotein (P-gp) efflux transport via P-gp ATPase inhibition. The current in vitro research using Caco-2 cells was conducted to further explore the P-gp ATPase inhibition mechanism. Using a monoclonal CD243 P-gp antibody shift assay (UIC2), we probed P-gp conformational changes induced via TPGS 1000. In the presence of TPGS 1000, UIC2 binding was slightly decreased. TPGS 1000 does not appear to be a P-gp substrate, nor does it function as a competitive inhibitor in P-gp substrate efflux transport. The reduction in UIC2 binding with TPGS 1000 was markedly weaker than with orthovanadate, data ruling out trapping P-gp in a transition state by direct interaction with one or both of the P-gp ATP nucleotide binding domains. An intracellular ATP depletion mechanism could be ruled out in the UIC2 assay, and by monitoring intracellular ATP levels in the presence of TPGS 1000. Indicating slow distribution of TPGS 1000 into the membrane, and in agreement with an intramembranal or intracellular side of action, Caco-2 cell monolayer experiments preincubated with TPGS 1000 produce stronger substrate inhibitory activity than those conducted by direct substrate and surfactant coapplication.

Upon hypoxia, erythrocytes utilize hemoglobin (Hb) to trigger activation of glycolysis through its interaction with band 3. This process contributes to maintenance of ATP, a portion of which is released extracellularly to trigger endothelium-dependent vasorelaxation. However, whether the ATP release results either from metabolic activation of the cells secondarily or from direct regulation of the gating through Hb allostery remains unknown. This study aimed to examine if stabilization of T-state Hb could induce steady-state and hypoxia-induced alterations in glycolysis and the ATP release from erythrocytes. Treatment of deoxygenated erythrocytes with a nitric oxide (NO) donor generated alpha-NO Hb that is stabilized T-state allostery. Under these circumstances, the release of ATP was significantly elevated even under normoxia and not further enhanced upon hypoxia. These events did not coincide with activation of glycolysis of the cells, so far as judged by the fact that intracellular ATP was significantly decreased by the NO treatment. Collectively, the present study suggests that hypoxia-induced ATP release is triggered through mechanisms involving R-T transition of Hb, and the gating process might occur irrespective of hypoxia-responsive regulation of glycolysis.

The 70 kDa heat shock proteins (Hsp70s) play important roles in preventing the misfolding of proteins and repairing damage under stress by coupling ATP binding and hydrolysis to protein substrate release and binding, respectively. ATP binding is believed to induce closing of the Hsp70 nucleotide binding domain (NBD) around the nucleotide. We report here a combined computational-experimental study of this open-closed transition. All-atom molecular dynamics simulations were performed for isolated open state NBDs with and without bound ATP. The nucleotide-free NBD samples a wide range of open configurations exhibiting flexible rearrangements of its four subdomains (IA-IIB). In contrast, the ATP-bound Hsp70 NBD closes to a range of configurations that is substantially more closed than the conformation observed in crystals of ATP-complexed NBDs. The close approach of subdomains IB and IIB observed in the simulations results in a strong coordination of the fluorescence probe Trp90 of IB with Arg261 of IIB, a feature not seen in the crystal structures. To determine if this computationally observed conformation occurs in solution, we constructed an R261A mutant. The mutation was found to increase the K(m) and k(cat) for ATP and to significantly reduce the extent of the fluorescence quench observed upon ATP binding. Our results thus account for the previously unexplained ATP-driven change in Trp90 fluorescence seen in the isolated NBD.

CaMKII, a major mediator of synaptic plasticity, forms extra-synaptic clusters under ischemic conditions. This study further supports self-aggregation of CaMKII holoenzymes as the underlying mechanism. Aggregation in vitro was promoted by mimicking ischemic conditions: low pH (6.8 or less), Ca(2+) (and calmodulin), and low ATP and/or high ADP concentration. Mutational analysis showed that high ATP prevented aggregation by a mechanism involving T286 auto-phosphorylation, and indicated requirement for nucleotide binding but not auto-phosphorylation also for extra-synaptic clustering within neurons. These results clarify a previously apparent paradox in the nucleotide and phosphorylation requirement of aggregation, and support a mechanism that involves inter-holoenzyme T286-region/T-site interaction.

Conformational changes of the Na(+)/K(+)-ATPase isolated large cytoplasmic segment connecting transmembrane helices M4 and M5 (C45) induced by the interaction with enzyme ligands (i.e. Mg(2+) and/or ATP) were investigated by means of the intrinsic tryptophan fluorescence measurement and molecular dynamic simulations. Our data revealed that this model system consisting of only two domains retained the ability to adopt open or closed conformation, i.e. behavior, which is expected from the crystal structures of relative Ca(2+)-ATPase from sarco(endo)plasmic reticulum for the corresponding part of the entire enzyme. Our data revealed that the C45 is found in the closed conformation in the absence of any ligand, in the presence of Mg(2+) only, or in the simultaneous presence of Mg(2+) and ATP. Binding of the ATP alone (i.e. in the absence of Mg(2+)) induced open conformation of the C45. The fact that the transmembrane part of the enzyme was absent in our experiments suggested that the observed conformational changes are consequences only of the interaction with ATP or Mg(2+) and may not be related to the transported cations binding/release, as generally believed. Our data are consistent with the model, where ATP binding to the low-affinity site induces conformational change of the cytoplasmic part of the enzyme, traditionally attributed to E2-->E1 transition, and subsequent Mg(2+) binding to the enzyme-ATP complex induces in turn conformational change traditionally attributed to E1-->E2 transition.

P-Glycoprotein (P-gp, ABCB1) transports a variety of structurally unrelated cytotoxic compounds out of the cell. Each homologous half of P-gp has a transmembrane (TM) domain containing six TM segments and a nucleotide-binding domain (NBD) and is joined by a linker region. It has been postulated that binding of two ATP molecules at the NBD interface to form a "nucleotide sandwich" induces drug efflux by altering packing of the TM segments that make up the drug-binding pocket. To test if ATP binding alone could alter packing of the TM segments, we introduced catalytic carboxylate mutations (E556Q in NBD1 and E1201Q in NBD2) into double-cysteine mutants that exhibited ATP-dependent cross-linking so that the mutants could bind but not hydrolyze ATP. It was found that ATP binding alone could alter disulfide cross-linking between the TM segments. For example, ATP inhibited cross-linking of mutant L339C(TM6)/V982C(TM12)/E556Q(NBD1)/E1201Q(NBD2) but promoted cross-linking of mutant F343C(TM6)/V982C(TM12)/E556Q(NBD1)/E1201Q(NBD2). Cross-linking of some mutants, however, appeared to require ATP hydrolysis as introduction of the catalytic carboxylate mutations into mutant L332C(TM6)/L975C(TM12) inhibited ATP-dependent cross-linking. Cross-linking between cysteines in the TM segments also could be altered via introduction of a single catalytic carboxylate mutation into mutant L332C(TM6)/L975C(TM12) or by using the nonhydrolyzable ATP analogue, AMP.PNP. The results show that the TM segments are quite sensitive to changes within the ATP-binding sites because different conformations could be detected in the presence of ATP, AMP.PNP, during ATP hydrolysis or through mutation of the catalytic carboxylates.

ATP-sensitive K+ channels (K(ATP) channels) are metabolic sensors formed by association of a K+ channel, Kir6, and an ATP-binding cassette (ABC) protein, SUR, which allosterically regulates channel gating in response to nucleotides and pharmaceutical openers and blockers. How nucleotide binding to SUR translates into modulation of Kir6 gating remains largely unknown. To address this issue, we have used a novel conformational KATP channel inhibitor, rhodamine 123 (Rho123) which targets the Kir6 subunit in a SUR-dependent manner. Rho123 blocked SUR-less Kir6.2 channels with an affinity of approximately 1 microM, regardless of the presence of nucleotides, but it had no effect on channels formed by the association of Kir6.2 and the N-terminal transmembrane domain TMD0 of SUR. Rho123 blocked SUR + Kir6.2 channels with the same affinity as Kir6.2 but this effect was antagonized by ATP. Protection from Rho123 block by ATP was due to direct binding of ATP to SUR and did not entail hydrolysis because it was not mimicked by AMP, did not require Mg2+ and was reduced by mutations in the nucleotide-binding domains of SUR. These results suggest that Rho123 binds at the TMD0-Kir6.2 interface and that binding of ATP to SUR triggers a change in the structure of the contact zone between Kir6.2 and domain TMD0 of SUR that causes masking of the Rho123 site on Kir6.2.

DNA damage-dependent signaling by the DNA mismatch repair (MMR) system is thought to mediate cytotoxicity of the anti-tumor drug cisplatin through molecular mechanisms that could differ from those required for normal mismatch repair. The present study investigated whether ATP-dependent biochemical properties of Escherichia coli MutS protein differ when the protein interacts with a DNA oligonucleotide containing a GT mismatch versus a unique site specifically placed cisplatin compound lesion, a cisplatin 1,2-d(GpG) intrastrand cross-link with a mispaired thymine opposite the 3' platinated guanine. MutS exhibited substantial affinity for this compound lesion in hydrolytic and in non-hydrolytic conditions of ATP, contrasting with the normal nucleotide inhibition effect of mispair binding. The cisplatin compound lesion was also shown to stimulate poorly MutS ATPase activity to approach the hydrolysis rate induced by nonspecific DNA. Moreover, MutS undergoes distinct conformation changes in the presence of the compound lesion and ATP under hydrolytic conditions as shown by limited proteolysis. In the absence of MutS, the cisplatin compound lesion was shown to induce a 39 degrees rigid bending of the DNA double helix contrasting with an unbent state for DNA containing a GT mispair. Furthermore, an unbent DNA substrate containing a monofunctional adduct mimicking a cisplatin residue failed to form a persistent nucleoprotein complex with MutS in the presence of adenine nucleotide. We propose that DNA bending could play a role in MutS biochemical modulations induced by a compound lesion and that cisplatin DNA damage signaling by the MMR system could be modulated in a direct mode.

The human P-glycoprotein (Pgp, ABCB1) is an ATP-dependent efflux pump for structurally unrelated hydrophobic compounds, conferring simultaneous resistance to and restricting bioavailability of several anticancer and antimicrobial agents. Drug transport by Pgp requires a coordinated communication between its substrate binding/translocating pathway (substrate site) and the nucleotide binding domains (NBDs or ATP sites). In this study, we demonstrate that certain thioxanthene-based Pgp modulators, such as cis-(Z)-flupentixol and its closely related analogues, effectively disrupt molecular cross talk between the substrate, and the ATP, sites without affecting the basic functional aspects of the two domains, such as substrate recognition, binding, and hydrolysis of ATP and dissociation of ADP following ATP hydrolysis. The allosteric modulator cis-(Z)-flupentixol has no effect on [alpha-(32)P]-8-azido-ATP binding to Pgp under nonhydrolytic conditions or on the K(m) for ATP during ATP hydrolysis. Both hydrolysis of ATP and vanadate-induced [alpha-(32)P]-8-azido-ADP trapping (following [alpha-(32)P]-8-azido-ATP breakdown) by Pgp are stimulated by the modulator. However, the ability of Pgp substrates (such as prazosin) to stimulate ATP hydrolysis and facilitate vanadate-induced trapping of [alpha-(32)P]-8-azido-ADP is substantially affected in the presence of cis-(Z)-flupentixol. Substrate recognition by Pgp as determined by [(125)I]iodoarylazidoprazosin ([(125)I]IAAP) binding both in the presence and in the absence of ATP is facilitated by the modulator, whereas substrate dissociation in response to vanadate trapping is considerably affected in its presence. In the Pgp F983A mutant, which is impaired in modulation by cis-(Z)-flupentixol, the modulator has a minimal effect on substrate-stimulated ATP hydrolysis as well as on substrate dissociation coupled to vanadate trapping. Finally, cis-(Z)-flupentixol has no effect on dissociation of [alpha-(32)P]-8-azido-ADP (or ADP) from vanadate-trapped Pgp, which is essential for subsequent rounds of ATP hydrolysis. Taken together, our results demonstrate a distinct mechanism of Pgp modulation that involves allosteric disruption of molecular cross talk between the substrate, and the ATP, sites without any direct interference with their individual functions.

An increasing body of evidence ascribes to misfolded forms of cytochrome c (cyt c) a role in pathophysiological events such as apoptosis and disease. Here, we examine the conformational changes induced by lipid binding to horse heart cyt c at pH 7 and study the ability of ATP (and other nucleotides) to refold several forms of unfolded cyt c such as oleic acid-bound cyt c, nicked cyt c, and acid denatured cyt c. The CD and fluorescence spectra demonstrate that cyt c unfolded by oleic acid has an intact secondary structure, and a disrupted tertiary structure and heme environment. Furthermore, evidence from the Soret CD, electronic absorption, and resonance Raman spectra indicates the presence of an equilibrium of at least two low-spin species having distinct heme-iron(III) coordination. As a whole, the data indicate that binding of cyt c to oleic acid leads to a partially unfolded conformation of the protein, resembling that typical of the molten globule state. Interestingly, the native conformation is almost fully recovered in the presence of ATP or dATP, while other nucleotides, such as GTP, are ineffective. Molecular modeling of ATP binding to cyt c and mutagenesis experiments show the interactions of phosphate groups with Lys88 and Arg91, with adenosine ring interaction with Glu62 explaining the unfavorable binding of GTP. The finding that ATP and dATP are unique among the nucleotides in being able to turn non-native states of cyt c back to native conformation is discussed in the light of cyt c involvement in cell apoptosis.

The Escherichia coli Rho protein uses the energy of ATP binding and hydrolysis to translocate along RNA and cause transcription termination. Using fluorescence stopped-flow kinetic studies, we have discerned the conformational changes in the Rho protein that occur upon nucleotide and nucleic acid binding. We show that the 2', (3')-O-[N-methylanthraniloyl] derivative of ATP (mant-ATP) is a good fluorescent substrate of Rho and is hydrolyzed with a K(m) comparable with that for ATP but a k(cat) five to six times slower than that for ATP. The kinetics of ATP and mant-ATP binding indicates that, in the absence of RNA, the Rho protein is structurally distinct from the Rho hexamer found when bound to RNA or DNA. In the absence of RNA, the nucleotide-binding rates are 50- to 70-fold slower, and the dissociation rates are 40- to 120-fold slower than the corresponding rates in the presence of RNA. We conclude that RNA or DNA binding to the primary nucleic acid binding sites causes conformational changes in the Rho hexamer that result in the opening of the subunit interfaces. Furthermore, the kinetic studies revealed a unique protein conformational change in the Rho.RNA complex upon ATP binding that is a result of RNA contacting the secondary nucleic acid binding sites in the central channel of the Rho ring. This conformational change seems to render the Rho ring competent in ATP hydrolysis and translocation.

Topoisomerase IV, a C(2)E(2) tetramer, is involved in the topological changes of DNA during replication. This enzyme is the target of antibacterial compounds, such as the coumarins, which target the ATP binding site in the ParE subunit, and the quinolones, which bind, outside the active site, to the quinolone resistance-determining region (QRDR). After site-directed and random mutagenesis, we found some mutations in the ATP binding site of ParE near the dimeric interface and outside the QRDR that conferred quinolone resistance to Streptococcus pneumoniae, a bacterial pathogen. Modeling of the N-terminal, 43-kDa ParE domain of S. pneumoniae revealed that the most frequent mutations affected conserved residues, among them His43 and His103, which are involved in the hydrogen bond network supporting ATP hydrolysis, and Met31, at the dimeric interface. All mutants showed a particular phenotype of resistance to fluoroquinolones and an increase in susceptibility to novobiocin. All mutations in ParE resulted in resistance only when associated with a mutation in the QRDR of the GyrA subunit. Our models of the closed and open conformations of the active site indicate that quinolones preferentially target topoisomerase IV of S. pneumoniae in its ATP-bound closed conformation.

We have used homology molecular modeling based on the srCaATPase E(2) conformation, pdb1kju, to predict side chains involved in docking the K(+) competitive inhibitor, SCH28080, to the H,K-ATPase. A model for SCH28080 binding between residues L809 and A335 in the same space utilized by omeprazole is proposed. We also describe modeling MgATP binding to the E(1) structure of the srATPase, pdb1eul, as a paradigm for the Na,K- and H,K-ATPases. The resulting model, E(1).MgATP, visualizes a conformation not yet available by crystallization and successfully predicts a range of published results, including backbone cleavages near V440 (N domain) and V712 (P domain) mediated by FeATP in the Na,K-ATPase. A separate model for MgATP docked to E(2) (pdb1kju) shows that access of the gamma phosphate to D351 is blocked by the A domain. The E(2). MgATP model explains FeATP-mediated cleavages of the Na,K-ATPase near V440 and E214 (A domain) and homologous results in the H,K-ATPase.

Binding ATP to tryptophanyl-tRNA synthetase (TrpRS) in a catalytically competent configuration for amino acid activation destabilizes the enzyme structure prior to forming the transition state. This conclusion follows from monitoring the titration of TrpRS with ATP by small angle solution X-ray scattering, enzyme activity, and crystal structures. ATP induces a significantly smaller radius of gyration at pH=7 with a transition midpoint at approximately 8mM. A non-reciprocal dependence of Trp and ATP dissociation constants on concentrations of the second substrate show that Trp binding enhances affinity for ATP, while the affinity for Trp falls with the square of the [ATP] over the same concentration range ( approximately 5mM) that induces the more compact conformation. Two distinct TrpRS:ATP structures have been solved, a high-affinity complex grown with 1mM ATP and a low-affinity complex grown at 10mM ATP. The former is isomorphous with unliganded TrpRS and the Trp complex from monoclinic crystals. Reacting groups of the two individually-bound substrates are separated by 6.7A. Although it lacks tryptophan, the low-affinity complex has a closed conformation similar to that observed in the presence of both ATP and Trp analogs such as indolmycin, and resembles a complex previously postulated to form in the closely-related TyrRS upon induced-fit active-site assembly, just prior to catalysis. Titration of TrpRS with ATP therefore successively produces structurally distinct high- and low-affinity ATP-bound states. The higher quality X-ray data for the closed ATP complex (2.2A) provide new structural details likely related to catalysis, including an extension of the KMSKS loop that engages the second lysine and serine residues, K195 and S196, with the alpha and gamma-phosphates; interactions of the K111 side-chain with the gamma-phosphate; and a water molecule bridging the consensus sequence residue T15 to the beta-phosphate. Induced-fit therefore strengthens active-site interactions with ATP, substantially intensifying the interaction of the KMSKS loop with the leaving PP(i) group. Formation of this conformation in the absence of a Trp analog implies that ATP is a key allosteric effector for TrpRS. The paradoxical requirement for high [ATP] implies that Gibbs binding free energy is stored in an unfavorable protein conformation and can then be recovered for useful purposes, including catalysis in the case of TrpRS.

Escherichia coli protease Lon (La) is an adenosine triphosphate (ATP)-regulated homo-oligomeric proteolytic complex responsible for the recognition and selective degradation of abnormal and unstable proteins. Each subunit of the protease Lon appears to consist of three functional domains: the C-terminal proteolytic containing a serine active site, the central displaying the ATPase activity, and the N-terminal with still obscure function. We have used limited proteolysis to probe the domain structure and nucleotide-induced conformational changes in the enzyme. Limited proteolysis of the native protease Lon generated a low number of stable fragments roughly corresponding to its functional domains. Conformational changes in the wild-type enzyme and its mutant forms in the presence or absence of adenine and guanine nucleotides were investigated by limited proteolysis. The nucleotide character was shown to play a key role for susceptibility of the protease Lon to limited proteolysis, in particular, for resistance of the ATPase functional domain. ATP and adenosine diphosphate displayed a protective effect of the ATPase domain of the enzyme. We suggest that these nucleotides induce conformational changes of the enzyme, transforming the ATPase domain from the most vulnerable part of the molecule into a spatially inaccessible one. Both limited proteolysis and autolysis demonstrate that the most stable part of the protease Lon molecule is its N-terminal region. Obvious resistance of the protease Lon C-terminus to proteolysis indicates that this region of the enzyme molecule including its substrate-binding and proteolytic domains has a well folded structure.

We have developed an angular refinement procedure incorporating correction for the microscope contrast transfer function, to determine cryoelectron microscopy (cryo-EM) structures of the Escherichia coli chaperonin GroEL in its apo and ATP-bound forms. This image reconstruction procedure is verified to 13-A resolution by comparison of the cryo-EM structure of unliganded GroEL with the crystal structure. Binding, encapsulation, and release of nonnative proteins by GroEL and its cochaperone GroES are controlled by the binding and hydrolysis of ATP. Seven ATP molecules bind cooperatively to one heptameric ring of GroEL. This binding causes long-range conformational changes that determine the orientations of remote substrate-binding sites, and it also determines the conformation of subunits in the opposite ring of GroEL, in a negatively cooperative mechanism. The conformation of GroEL-ATP was determined at approximately 15-A resolution. In one ring of GroEL-ATP, the apical (substrate-binding) domains are extremely disordered, consistent with the high mobility needed for them to achieve the 60 degrees elevation and 90 degrees twist of the GroES-bound state. Unexpectedly, ATP binding also increases the separation between the two rings, although the interring contacts are present in the density map., (Copyright 2001 Academic Press.)

Protein folding by chaperonins is powered by ATP binding and hydrolysis. ATPase activity drives the folding machine through a series of conformational rearrangements, extensively described for the group I chaperonin GroEL from Escherichia coli but still poorly understood for the group II chaperonins. The latter--archaeal thermosome and eukaryotic TRiC/CCT--function independently of a GroES-like cochaperonin and are proposed to rely on protrusions of their own apical domains for opening and closure in an ATP-controlled fashion. Here we use small-angle neutron scattering to analyze structural changes of the recombinant alpha-only and the native alphabeta-thermosome from Thermoplasma acidophilum upon their ATPase cycling in solution. We show that specific high-salt conditions, but not the presence of MgATP alone, induce formation of higher order thermosome aggregates. The mechanism of the open-closed transition of the thermosome is strongly temperature-dependent. ATP binding to the chaperonin appears to be a two-step process: at lower temperatures an open state of the ATP-thermosome is predominant, whereas heating to physiological temperatures induces its switching to a closed state. Our data reveal an analogy between the ATPase cycles of the two groups of chaperonins and enable us to put forward a model of thermosome action., (Copyright 2001 Academic Press.)

Current biochemical and structural studies on the conformational changes induced by the nature of nucleotide bound to the chaperonin containing testis complex polypeptide 1 (CCT) are examined to see how consistent the data are. This exercise suggests that the biochemical and structural data are in good agreement. CCT clearly appears as a folding nano-machine fueled by ATP. A careful comparison of the biochemical and structural data, however, highlights a number of points that remain to be carefully documented in order to better understand the nature of the conformational changes in CCT that yield folded target proteins. Special effort should be made to clearly answer the points listed at the end of this review in order to obtain the dynamic sequence of events yielding folded proteins in the eukaryotic cytoplasm similar to what has been obtained for prokaryotes., (Copyright 2001 Academic Press.)

The nonstructural protein NSP2 is a component of the rotavirus replication machinery and binds single-stranded RNA cooperatively, with high affinity, and independent of sequence. Recently, NSP2 has been shown to form multimers and to possess an NTPase activity, but its precise function remains unclear. In the present study, we have characterized the solution structure of recombinant NSP2 by velocity and equilibrium ultracentrifugation, dynamic light scattering, and circular dichroism spectroscopy. We found that NSP2 exists as an octamer, which is functional in the binding of RNA and ADP. In the presence of magnesium, a partial dissociation of the octamer into smaller oligomers was observed. This was reversed by binding of ADP and RNA. We observed an increased sedimentation rate in the presence of ADP and a nonhydrolyzable ATP analogue, which suggests a change toward a significantly more compact octameric conformation. The secondary structure of NSP2 showed a high fraction of beta-sheet, with small changes induced by magnesium that were reversed in the presence of RNA. That NSP2 can exist in different conformations lends support to the previously proposed hypothesis (Taraporewala, Z., Chen, D., and Patton, J. T. (1999) J. Virol. 73, 9934-9943) of its function as a molecular motor involved in the packaging of viral mRNA.

To investigate ATP as a potential factor in the regulation of brain glutamate decarboxylase (GAD), the impact of ATP on the enzyme conformation and holoenzyme formation was investigated. ATP at 100 microM quenches fluorescence emission intensity of the holoenzyme of GAD (holoGAD) by 18% after a correction for the inner filter effect and enhances fluorescence steady-state polarization from 0.158 to 0. 183 when excited at 280 or 295 nm. These findings suggest that ATP moderately changes the microenvironment of one or more tryptophan or tyrosine residues in holoGAD and alters these residues from a more mobile state to a less mobile one. A moderate ATP-induced conformational change in holoGAD is also supported by the observations that ATP increases the thermal denaturation temperature of holoGAD by 2 degrees C, as derived from temperature-dependent fluorescence spectra, and decreases the alpha-helical content of holoGAD by 8-10%, as determined by circular dichroism. Moreover, ATP does not affect the keto-enol tautomerization of holoGAD and has little or no direct effect on its activity, implying that the ATP interacting domain in holoGAD is not at the active site. Kinetics studies, as demonstrated by stopped-flow fluorescence and UV/visible spectroscopy, demonstrate that formation of holoGAD involves two steps: a fast reaction forming an apoGAD-cofactor intermediate complex, followed by a slow reaction involving the conformational change in the intermediate complex. ATP reduces the rate constant of the fast step to one-third and decreases the rate of the slow step and the intermediate complex formation constant to 60% of their original values. The present data suggest that ATP may regulate the interconversion between apoGAD and holoGAD by interacting with apoGAD rather than holoGAD. By slowing down the rate of intermediate complex formation, ATP reduces the amount of holoGAD formed., (Copyright 2000 Academic Press.)

ATP hydrolysis by MutS homologues is required for the function of these proteins in mismatch repair. However, the function of ATP hydrolysis in the repair reaction is not very clear. We have examined the role of ATP hydrolysis in oligomerization of Thermus aquaticus (Taq) MutS protein in solution. Analytical gel filtration and cross-linking of MutS protein with disuccinimidyl suburate suggest that TaqMutS is a dimer in the presence of ATP. ATP binding and hydrolysis by TaqMutS reduces the heteroduplex-DNA binding by the protein. Using limited proteolysis we detected extensive conformational changes of the TaqMutS protein in the presence of ATP and heteroduplex DNA. Heteroduplex-DNA binding is necessary for the observed conformational changes since F39A mutant protein defective in DNA binding does not display ATP-induced conformational changes. The implications of the observed conformational changes in the MutS protein are discussed with respect to two different models proposed for the role of ATP hydrolysis by MutS in DNA mismatch repair.

It has been known for some time that ATP inhibits the DNA relaxation activity of human DNA topoisomerase I. However, the underlying mechanism of this inhibitory effect remains largely unknown. Using filter binding assays, the binding of human DNA topoisomerase I to DNA was decreased in the presence of ATP. This result suggests that the inhibition of DNA relaxation activity of human DNA topoisomerase I by ATP is at the binding step rather than at the nicking or resealing step. DNA topoisomerase I cleavage assay further supports this notion. ATP-agarose binding and UV cross-linking assays also demonstrate that ATP directly and specifically binds human DNA topoisomerase I. To address whether the ATP binding results in conformational changes in human DNA topoisomerase I, various proteases were employed for detecting potential protein conformational changes. Our results indicated that the proteolytic susceptibilities of trypsin and chymotrypsin were altered in the presence of ATP. The result suggests that the conformation of human DNA topoisomerase I was altered upon ATP binding. In addition, the binding between ATP and human DNA topoisomerase I was also reduced by increasing concentrations of DNA. Our data suggests that human DNA topoisomerase I exhibits at least two incompatible conformations. One conformation is in the form of a topoisomerase I-ATP complex, which inhibits DNA relaxation activity of human DNA topoisomerase I, and the other, a topoisomerase I-DNA complex, which exerts DNA relaxation activity. Our studies identify the role of ATP in the regulation of human DNA topoisomerase I and provide a substantial implication of how human DNA topoisomerase I compromises its versatile functions.

To define the role of the Cys residues in the ATP-dependent HslVU protease, mutagenesis was performed to replace either Cys261 or Cys287 in HslU with Val and Cys159 in HslV with Ser or Ala. Whereas HslU/C261V could hydrolyze ATP and support the ATP-dependent proteolytic activity of HslV as well as the wild-type HslU, HslU/C287V could not hydrolyze ATP. Nevertheless, HslU/C287V could support the HslV-mediated proteolysis by forming the HslVU complex in the presence of ATP but not its absence, indicating that ATP binding but not its hydrolysis is essential for proteolysis. Whereas treatment of N-ethylmaleimide (NEM) resulted in dissociation of the oligomeric HslU into monomers, the C261V mutation, but not C287V, prevented the NEM effect. These results suggest that Cys261 is involved in oligomerization and that Cys287 is related to the ATPase function of HslU. NEM also dissociated the dodecameric HslV into monomers, and this effect could be prevented by either the C159S or C159A mutation, suggesting the involvement of Cys159 in oligomerization of HslV. Moreover, either mutation abolished both the basal and HslU-activated proteolytic activity of HslV and its ability to activate the HslU ATPase and to form the HslVU complex, indicating that Cys159 is essential for the proteolytic activity of HslV and its interaction with HslU. These results suggest that the Cys residues play an important role in maintaining the structure and function of the HslVU protease.

The eukaryotic cytosolic chaperonins are large heterooligomeric complexes with a cylindrical shape, resembling that of the homooligomeric bacterial counterpart, GroEL. In analogy to GroEL, changes in shape of the cytosolic chaperonin have been detected in the presence of MgATP using electron microscopy but, in contrast to the nucleotide-induced conformational changes in GroEL, no details are available about the specific nature of these changes. The present study identifies the structural regions of the cytosolic chaperonin that undergo conformational changes when MgATP binds to the nucleotide binding domains. It is shown that limited proteolysis with trypsin in the absence of MgATP cleaves each of the eight subunits approximately in half, generating two fragments of approximately 30 kDa. Using mass spectrometry (MS) and N-terminal sequence analysis, the cleavage is found to occur in a narrow span of the amino acid sequence, corresponding to the peptide binding regions of GroEL and to the helical protrusion, recently identified in the structure of the substrate binding domain of the archeal group II chaperonin. This proteolytic cleavage is prevented by MgATP but not by ATP in the absence of magnesium, ATP analogs (MgATPyS and MgAMP-PNP) or MgADP. These results suggest that, in analogy to GroEL, binding of MgATP to the nucleotide binding domains of the cytosolic chaperonin induces long range conformational changes in the polypeptide binding domains. It is postulated that despite their different subunit composition and substrate specificity, group I and group II chaperonins may share similar, functionally-important, conformational changes. Additional conformational changes are likely to involve a flexible helix-loop-helix motif, which is characteristic for all group II chaperonins.

The interaction between skeletal myosin subfragment 1 (S1) and filamentous actin was examined at various intermediate states of the actomyosin ATPase cycle by chemical cross-linking experiments. Reaction of the actin-S1 complex with 1-ethyl-3-[3-(dimethylamino)propyl]carbodiimide and N-hydroxysuccinimide generated products with molecular masses of 165 and 175 kDa, in which S1 loops of residues 626-647 and 567-578 were cross-linked independently to the N-terminal segment of residues 1-12 of one actin monomer, and of 265 kDa, in which the two loops were bound to the N termini of two adjacent monomers. In strong-binding complexes, i.e., without nucleotide or with ADP, S1 was sequentially cross-linked to one and then to two actin monomers. In the weak-binding complexes, two types of cross-linking pattern were observed. First, during steady-state hydrolysis of ATP or ATPgammaS at 20 degreesC, the cross-linking reaction gave rise to a small amount of unknown 200 kDa product. Second, in the presence of AMPPNP, ADP.BeFx, ADP.AlF4-, or ADP.VO43- or with S1 internally cross-linked by N,N'-p-phenylenedimaleimide, only the 265 kDa product was obtained. The presence of 200 mM salt inhibited cross-linking reactions in both weak- and strong-binding states, while it dissociated only weak-binding complexes. These results indicate that, in the weak-binding state populated with the ADP.Pi analogues, skeletal S1 interacts predominantly and with an apparent equal affinity with the N termini of two adjacent actin monomers, while these ionic contacts are much less significant in stabilizing the rigor actin-S1 complexes. They also suggest that the electrostatic actin-S1 interface is not influenced by the type of ADP.Pi analogue bound to the active site.

The conformation of ADPMg(II) bound at the active site of yeast hexokinase PI has been determined using transferred nuclear Overhauser effect spectroscopy (TRNOESY). We have measured the time dependent NOE buildup of all the proton pairs of ADP in enzyme. ADPMg(II) and enzyme. glucose.ADPMg(II) complexes at 500 MHz and 10 degrees C. The data have been analyzed using complete relaxation matrix approach to obtain various inter-proton distances. These distances were used as restraints in the molecular dynamics and energy minimization to obtain the conformation of the bound nucleotide. The results from these calculations suggest that in both the complexes, the nucleotide binds in an anti conformation with a glycosidic torsion angle chi = 55 +/- 5 degrees and 52 +/- 5 degrees in PI.ADPMg(II) and PI.glucose.ADPMg(II) complexes, respectively. However, the phase angle of pseudorotation (P) which defines the sugar pucker in the two complexes was found to be 99 degrees, corresponding to 0T1 for PI.ADPMg(II) complex and 69 degrees corresponding to 4T0 for PI.glucose.ADPMg(II) complex. The cleft closure conformational change in the enzyme induced by glucose seems to affect the conformation of the ribosyl moiety of the bound nucleotide.

Bile-salt-dependent lipase (BSDL) is secreted by the pancreas into the duodenum, where it catalyses the hydrolysis of dietary lipid esters on activation by bile salts. The secretion pathway of BSDL is comparable with that of other digestive enzymes produced by pancreatic acinar cells. However, in contrast with these other enzymes, BSDL is partly associated with endoplasmic reticulum membranes as part of a folding complex, including a Grp94-related protein to which BSDL is transiently linked. The release of BSDL from membranes occurs once its glycosylation is completed [Bruneau and Lombardo (1995) J. Biol. Chem. 270, 13524-13533]. In the present study, investigations concerning the mechanism of association/dissociation of BSDL with membranes of microsomes were performed. For this purpose the role of ATP and that of the possible phosphorylation of BSDL were examined. For the first time, it is shown that human pancreatic BSDL is phosphorylated, probably at a serine residue, during its transport within the acinar cell. The phosphorylation of BSDL is provoked by calphostin C, an inhibitor of protein kinase C. In the presence of 1-(isoquinolinesulphonyl)2-methylpiperazine, a non-specific inhibitor of serine/threonine protein kinase A, C or G, or of calcium chelator 1,2-bis(O-aminophenoxy)ethane-N,N,N', N'-tetra-acetic tetra(acetoxymethyl)ester, the phosphorylation of BSDL elicited by calphostin C is abolished. These data suggested that the phosphorylation of BSDL within human pancreatic microsomes is under the control of a cascade of protein kinases. We have also shown that the phosphorylation of BSDL appears to be involved in the release of the enzyme from microsome membranes. Nevertheless ATP, which modifies the conformation of BSDL, triggers this association, and an unhydrolysable ATP analogue was unable to promote it.

ATP hydrolysis by the cystic fibrosis transmembrane conductance regulator (CFTR) Cl- channel predicts that energy from hydrolysis might cause asymmetric transitions in the gating cycle. We found that 3-(N-morpholino)propanesulfonic acid (MOPS) blocked the open channel by binding to a site 50% of the way through the electrical field. Block by MOPS revealed two distinct states, O1 and O2, which showed a strong asymmetry during bursts of activity; the first opening in a burst was in the O1 state and the last was in the O2 state. Addition of a nonhydrolyzable nucleoside triphosphate prevented the transition to the O2 state and prolonged the O1 state. These data indicate that ATP hydrolysis by the nucleotide-binding domains drives a series of asymmetric transitions in the gating cycle. They also indicate that ATP hydrolysis changes the conformation of the pore, thereby altering MOPS binding.

Receptor dimerization is critical for signaling by the epidermal growth factor receptor (EGFR) tyrosine kinase. This occurs after binding of the receptor's extracellular domain by ligand or bivalent antibodies. The role of other receptor domains in dimerization is less clear, and there are no examples of dimers induced by direct perturbation of the EGFR kinase domain. Submicromolar concentrations of AG-1478 and AG-1517, quinazolines specific for inhibition of the EGFR kinase, induced reversible receptor dimerization in vitro and in intact A431 cells. Consistent with the inhibitory effect of quinazolines on receptor kinase activity, the dimers formed lacked a detectable Tyr(P) signal. Quinazoline-induced EGFR dimerization was abrogated in vitro by ATP and the ATP analog adenyl-5'-yl imidodiphosphate. Receptors with a single-point mutation in the ATP binding site as well as wild-type EGFR with a covalent modification of the ATP site failed to dimerize in response to AG-1478 and AG-1517. These data suggest that EGFR dimerization can be induced by the interaction of quinazolines at the ATP site in the absence of receptor ligand binding. In SKBR-3 cells, the quinazolines induced the formation of inactive EGFR/ErbB-2 heterodimers, potentially sequestering ErbB-2 from interacting with other coreceptors of the ErbB family. Structural studies of the quinazoline interaction with the EGFR tyrosine kinase domain should allow for an analysis of receptor-specific chemical features required for binding to the ATP site and disruption of signaling, a strategy that can be perhaps applied to other tumor cell receptor systems.

Nicotinic acid phosphoribosyltransferase (NAPRTase;EC 2.4.2.11) couples stoichiometric ATP hydrolysis with formation of nicotinate mononucleotide (NAMN) from nicotinic acid and alpha-D-5-phosphoribosyl 1-pyrophosphate (PRPP). Trypsin rapidly inactivated the ATPase and NAMN synthesis activities of NAPRTase in parallel, with cleavages at Arg-384 and Lys-374 of the 399-residue protein. ATP and PRPP each provided protection against tryptic cleavage. Limited chymotryptic proteolysis of NAPRTase exhibited very similar behavior, with specific cleavage at Phe-382 and protection by substrates. Results suggest that a solvent-exposed loop encompassing Lys-374, Phe-382, and Arg-384 is protected by ATP- or PRPP-induced conformational changes. The ability of ATP to protect even under conditions in which enzyme phosphorylation was prevented by EDTA provides evidence for a distinct ATP-induced protein conformation that acts as an intermediate in energy coupling.

ATP and luciferin are not only substrates of firefly luciferase, but can, in addition, modulate its activity. High concentrations of luciferin induce a conformational change of the enzyme that temporarily reduces the catalytic rate. Re-activation takes approx. 20 min and is independent of variation in the concentration of enzyme or ATP, but lengthens with increasing luciferin concentration. High concentrations of albumin reduce this luciferin effect. The kinetic properties of firefly luciferase determined from initial rates and at steady state after 1 min of catalysis have been analysed according to Michaelis-Menten kinetics. There is only one active site for each of the substrates. At steady state the Km and Vmax. values for both substrates are reduced in an uncompetitive manner. Hyperbolic Lineweaver-Burk plots indicate an activation by ATP probably by binding to an allosteric site. A model is presented which incorporates luciferin induced de- and re-activation effects. Experimental conditions to avoid the regulatory effects of substrates during ATP monitoring are proposed.

Cys-674 of the sarcoplasmic reticulum Ca(2+)-ATPase was labeled with N-acetyl-N'-(5-sulfo-1-naphthyl)ethylenediamine without a loss of the catalytic activity. The steady-state fluorescence of the label decreased with increasing concentration of Mg.ATP, Mn.ATP, or Ca.ATP in the presence of Ca2+ and 0.1 M KCl at 0 degree C, pH 7.0. The maximum extent of the decrease was 23%. The Mg.ATP-, Mn.ATP-, or Ca.ATP-induced fluorescence drop was also determined by the stopped-flow spectrofluorometry. The Ca.ATP-induced fluorescence drop manifested a biphasic time course. The first phase reflects the conformational change which was previously shown [Suzuki, H., Obara, M., Kuwayama, H., & Kanazawa, T. (1987) J. Biol. Chem. 262, 15448-15456] to occur upon formation of the calcium-enzyme-substrate complex. The amplitude of the second phase was 2.5% of the fluorescence before addition of Ca.ATP. This phase of the fluorescence drop coincided with the phosphoenzyme formation, which was determined by the continuous flow-rapid quenching method. The phosphoenzyme formed was largely sensitive to ADP. When adenosine 5'-(beta,gamma-methylenetriphosphate) (a nonhydrolyzable ATP analog incapable of phosphorylating the enzyme) was added in the presence of 5 mM CaCl2 without added MgCl2 the steady-state fluorescence decreased rapidly by 20%. However, this drop lacked the second phase. When phosphoenzyme isomerization from the ADP-sensitive form to the ADP-insensitive form was prevented by the N-ethylmaleimide treatment, the second phase of the Ca.ATP-induced fluorescence drop again coincided with the phosphoenzyme formation.(ABSTRACT TRUNCATED AT 250 WORDS)

Small angle x-ray scattering experiments have been carried out on the purified iron proteins of nitrogenase from wild-type Azotobacter vinelandii and from a Nif- mutant strain, A. vinelandii UW91 (which has an A157S mutation). This study was designed to investigate the influence of MgATP and MgADP binding on the protein structure in solution. For the wild-type protein, the binding of MgATP induces a significant conformational change that is observed as a decrease of about 2.0 A in the radius of gyration. In contrast, the binding of MgADP to the wild-type iron protein does not detectably affect the radius of gyration. In the absence of nucleotides, the radius of gyration for the UW91 mutant is indistinguishable from that of the wild-type. However, unlike for the wild-type protein, the radius of gyration of the UW91 iron protein is unaffected by the addition of MgATP. We have previously shown that the UW91 iron protein has a normal [4Fe-4S] cluster and MgATP binding ability but that it is completely blocked for electron transfer and MgATP hydrolysis (Gavini, N., and Burgess, B. K. (1992) J. Biol. Chem. 267, 21179-21186). These x-ray scattering measurements suggest that a conformation different from that of the native state is therefore required for the iron protein to perform electron transfer to the MoFe protein. These results also support the hypothesis that Ala-157 is crucial for the iron protein to establish the electron-transfer-favored conformation induced by MgATP binding.

Ligand-induced allosteric changes in proteins are important in their cellular functions and regulation, and both concerted and sequential examples are known. The distinction has entailed elaborate analysis, however, and only a few systems have been unequivocally analysed. We have investigated the coupling between ATP usage and DNA transport by type II DNA topoisomerases, and one key question concerning allostery in these dyadic enzymes is whether ATP binding to one protomer can induce a concerted conformational change in the entire enzyme. Here we use an enzyme with one immunotagged subunit defective in ATP binding and one wild-type subunit to show that it can. Our approach should be generally applicable in the study of allostery and communication between members of a macromolecular assembly.

The 90-kDa heat shock protein (hsp90) is a well conserved, abundant cytosolic protein believed to be a "chaperone" of most steroid receptors. We have recently demonstrated that hsp90 has an ATP-binding site and autophosphorylating activity (Csermely, P., and Kahn, C. R. (1991) J. Biol. Chem. 266, 4943-4950). Circular dichroism analysis of highly purified hsp90 from rat liver shows that ATP induces an increase of beta-pleated sheet content of hsp90. Vanadate, molybdate, and heat treatment at 56 degrees C induce a similar change in the circular dichroism spectrum. Fourier transformed infrared spectroscopy reveals an ATP-induced increase in the interchain interactions of the 90-kDa heat shock protein due to an increase in its beta-pleated sheet content. In further studies we found that ATP: 1) decreases the tryptophan fluorescence of hsp90 by 11.6 +/- 1.9%; 2) increases the hydrophobic character of the protein as determined by its distribution between an aqueous phase and phenyl-Sepharose; and 3) renders hsp90 less susceptible to tryptic digestion. Our results suggest that hsp90 undergoes an "open-->closed" conformational change after the addition of ATP, analogous in many respects to the similar changes of the DnaK protein, the immunoglobulin heavy chain binding protein (BiP/GRP78), and hsp70. The ATP-induced conformational change of hsp90 may be important in regulating its association with steroid receptors and other cellular proteins.

Membrane-bound Na+/K(+)-ATPase purified from dog kidney was solubilized with octaethylene glycol dodecyl ether (C12E8), and the resultant solubilized enzyme was chromatographed on a TSKgel G4000SWXL or G3000SWXL column equilibrated with elution buffers containing various ligands affecting oligomerization of the enzyme. Weight-averaged molecular weight (Mw) values for the main protein components eluted were estimated by low-angle laser light-scattering photometry. With increasing concentration of C12E8 included in the elution buffer from 0.1 to 5 mg/ml, the Mw decreased from 230,000 to 153,000, indicating that C12E8 induced dissociation of the enzyme. In contrast, the Mw of the protein component increased up to 1.44.10(6) as the concentration of phosphatidylserine (PS) added to the elution buffer containing a fixed concentration of 0.3 mg/ml C12E8 was increased to 120 micrograms/ml. The association and/or aggregation were reversible by removal of the PS by rechromatography. Addition of PS to the elution buffer also allowed the solubilized enzyme to exhibit ATPase activity comparable to that of the membrane-bound enzyme during passage through the column. This was also the case with phosphatidylglycerol (PG) and phosphatidylinositol, but not with phosphatidylcholine or phosphatidylethanolamine. The specific refractive index increment (dn/dcp) of the solubilized enzyme was increased by addition of exogenous PG or PS, strongly suggesting that the phospholipid became bound to the enzyme, and that it induced association of the enzyme. The association induced by PS was inhibited by ATP and ADP, but not AMP. The concentrations for half-maximal inhibition were 0.44 mM for ATP and 0.88 mM for ADP. The PS-induced associated enzyme isolated by chromatography in the presence of 120 micrograms/ml PS was dissociated by ATP with K0.5 of 0.16 mM. The dissociating effect of C12E8, ATP and ADP and the associating effect of PS on the solubilized enzyme are consistent with the reports that C12E8 mimics the effect of regulatory ATP at the low-affinity site on the conformational transition from E2 to E1, and that phospholipids are essential for the reverse transition from E1 to E2. The results can be explained by assuming that the enzyme takes the form of a loosely associated diprotomer in the E1 state and a tightly associated one in the E2 state.

A change in the conformation of the active site of scallop myosin under the influence of regulatory amounts of Ca2+ has been identified by use of the ADP photoaffinity analog 2-[(4-azido-2-nitrophenyl)amino]ethyl diphosphate (NANDP). NANDP, trapped at the active site with Mn2+ and vanadate, photolabeled preferentially Arg-128 of the heavy chain in the absence of added Mg2+ and Ca2+ [Kerwin, B. & Yount, R. (1992) Bioconjugate Chem. 3, 328-336]. However, addition of 2 mM Mg2+ and regulatory amounts of Ca2+ (0.01-1 microM) shifted the predominant labeling to Cys-198 of the heavy chain in a Ca(2+)-dependent manner. This Ca(2+)-dependent change in the photolabeling pattern was absent when the regulatory light chains were removed or when the unregulated head (subfragment 1) was examined under similar conditions. These results demonstrate that both Arg-128 and Cys-198 are part of the purine binding site which undergoes a conformational change in response to Ca2+ binding to the regulatory domain.