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Down-regulation of Ras signalling is mediated by specific GTPase-activating proteins (GAPs), which stimulate the very slow GTPase reaction of Ras by 10(5)-fold. The basic features of the GAP activity involve the stabilisation of both switch regions of Ras in the transition state, and the insertion of an arginine finger. In the case of oncogenic Ras mutations, the features of the active site are disturbed. To understand these features in more detail, we have investigated the effects of oncogenic mutations of Ras and compared the GAP-stimulated GTPase reaction with the ability to form GAP-mediated aluminium or beryllium fluoride complexes. In general we find a correlation between the size of the amino acid at position 12, the GTPase activity and ability to form aluminium fluoride complexes. While Gly12 is very sensitive to even the smallest possible structural change, Gly13 is much less sensitive to steric hindrance, but is sensitive to charge. Oncogenic mutants of Ras defective in the GTPase activity can however form ground-state GppNHp complexes with GAP, which can be mimicked by beryllium fluoride binding. We show that beryllium fluoride complexes are less sensitive to structural changes and report on a state close to but different from the ground state of the GAP-stimulated GTPase reaction.

A number of regulatory circuits in biological systems function through the exchange of phosphoryl groups from one protein to another. Spo0F and Spo0B are components of a phosphorelay that control sporulation in the bacterium Bacillus subtilis through the exchange of a phosphoryl group. Using beryllofluoride as a mimic for phosphorylation, we trapped the interaction of the phosphorylated Spo0F with Spo0B in the crystal lattice. The transition state of phosphoryl transfer continues to be a highly debated issue, as to whether it is associative or dissociative in nature. The geometry of Spo0F binding to Spo0B favors an associative mechanism for phosphoryl transfer. In order to visualize the autophosphorylation of the histidine kinase, KinA, and the subsequent phosphoryl transfer to Spo0F, we generated in silico models representing these reaction steps.

The structure of bovine F1-ATPase inhibited with ADP and beryllium fluoride at 2.0 angstroms resolution contains two ADP.BeF3- complexes mimicking ATP, bound in the catalytic sites of the beta(TP) and beta(DP) subunits. Except for a 1 angstrom shift in the guanidinium of alphaArg373, the conformations of catalytic side chains are very similar in both sites. However, the ordered water molecule that carries out nucleophilic attack on the gamma-phosphate of ATP during hydrolysis is 2.6 angstroms from the beryllium in the beta(DP) subunit and 3.8 angstroms away in the beta(TP) subunit, strongly indicating that the beta(DP) subunit is the catalytically active conformation. In the structure of F1-ATPase with five bound ADP molecules (three in alpha-subunits, one each in the beta(TP) and beta(DP) subunits), which has also been determined, the conformation of alphaArg373 suggests that it senses the presence (or absence) of the gamma-phosphate of ATP. Two catalytic schemes are discussed concerning the various structures of bovine F1-ATPase.

To understand the mechanism of a functionally important ATP-induced allosteric transition of GroEL, we have studied the effect of a series of metal fluoride-ADP complexes and vanadate-ADP on GroEL by kinetic fluorescence measurement of pyrene-labeled GroEL and by small-angle X-ray scattering measurement of wild-type GroEL. The metal fluorides and vanadate, complexed with ADP, are known to mimic the gamma-phosphate group of ATP, but they differ in geometry and size; it is expected that these compounds will be useful for investigating the strikingly high specificity of GroEL for ATP that enables the induction of the allosteric transition. The kinetic fluorescence measurement revealed that aluminium, beryllium, and gallium ions, when complexed with the fluoride ion and ADP, induced a biphasic fluorescence change of pyrenyl GroEL, while scandium and vanadate ions did not induce any kinetically observed change in fluorescence. The burst phase and the first phase of the fluorescence kinetics were reversible, while the second phase and subsequent changes were irreversible. The dependence of the burst-phase and the first-phase fluorescence changes on the ADP concentration indicated that the burst phase represents non-cooperative nucleotide binding to GroEL, and that the first phase represents the allosteric transition of GroEL. Both the amplitude and the rate constant of the first phase of the fluorescence kinetics were well understood in terms of a kinetic allosteric model, which is a combination of transition state theory and the Monod-Wyman-Changeux allosteric model. From the kinetic allosteric model analysis, the relative free energy of the transition state in the metal fluoride-ADP-induced allosteric transition of GroEL was found to be larger than the corresponding free energy of the ATP-induced allosteric transition by more than 5.5kcal/mol. However, the X-ray scattering measurements indicated that the allosteric state induced by these metal fluoride-ADP complexes is structurally equivalent to the allosteric state induced by ATP. These results suggested that both the size and coordination geometry of gamma-phosphate (and its analogs) are related to the allosteric transition of GroEL. It was therefore concluded that the tetrahedral geometry of gamma-phosphate (or its analogs) and the inter-atomic distance ( approximately 1.6A) between phosphorus (vanadium, or metal atom) and oxygen (or fluorine) are both important for inducing the allosteric transition of GroEL, leading to the high selectivity of GroEL for ATP about ligand adenine nucleotides, which function as the preferred allosteric ligand.

A Crystallogral structure is described for the Mg2+-BeF3--bound receiver domain of Sinorhizobium meliloti DctD bearing amino acid substitution E121K. Differences between the apo- and ligand-bound active sites are similar to those reported for other receiver domains. However, the off and on states of the DctD receiver domain are characterized by dramatically different dimeric structures, which supports the following hypothesis of signal transduction. In the off state, the receiver domain and coiled-coil linker form a dimer that inhibits oligomerization of the AAA+ ATPase domain. In this conformation, the receiver domain cannot be phosphorylated or bind Mg2+ and BeF3-. Instead, these modifications stabilize an alternative dimeric conformation that repositions the subunits by approximately 20 A, thus replacing the a4-b5-a5 interface with an a4-b5 interface. Reoriented receiver domains permit the ATPase domain to oligomerize and stimulate open complex formation by the s54 form of RNA polymerase. NtrC, which shares 38% sequence identity with DctD, works differently. Its activated receiver domain must facilitate oligomerization of its ATPase domain. Significant differences exist in the signaling surfaces of the DctD and NtrC receiver domains that may help explain how triggering the common two-component switch can variously regulate assembly of a AAA+ ATPase domain.

The CheY protein is the response regulator in bacterial chemotaxis. Phosphorylation of a conserved aspartyl residue induces structural changes that convert the protein from an inactive to an active state. The short half-life of the aspartyl-phosphate has precluded detailed structural analysis of the active protein. Persistent activation of Escherichia coli CheY was achieved by complexation with beryllofluoride (BeF(3)(-)) and the structure determined by NMR spectroscopy to a backbone r.m.s.d. of 0.58(+/-0.08) A. Formation of a hydrogen bond between the Thr87 OH group and an active site acceptor, presumably Asp57.BeF(3)(-), stabilizes a coupled rearrangement of highly conserved residues, Thr87 and Tyr106, along with displacement of beta4 and H4, to yield the active state. The coupled rearrangement may be a more general mechanism for activation of receiver domains., (Copyright 2000 Academic Press.)

Two-component systems, sensor kinase-response regulator pairs, dominate bacterial signal transduction. Regulation is exerted by phosphorylation of an Asp in receiver domains of response regulators. Lability of the acyl phosphate linkage has limited structure determination for the active, phosphorylated forms of receiver domains. As assessed by both functional and structural criteria, beryllofluoride yields an excellent analogue of aspartyl phosphate in response regulator NtrC, a bacterial enhancer-binding protein. Beryllofluoride also appears to activate the chemotaxis, sporulation, osmosensing, and nitrate/nitrite response regulators CheY, Spo0F, OmpR, and NarL, respectively. NMR spectroscopic studies indicate that beryllofluoride will facilitate both biochemical and structural characterization of the active forms of receiver domains.

Aluminum fluoride (AlF(4)(-)) inhibited phospholipase D (PLD) purified from cabbage in both PIP(2)-dependent and PIP(2)-independent assays, consistent with its previously observed effect on mammalian PLD. The possibility that AlF(4)(-) may exert this effect through its known phosphate-mimicking property was examined. Inorganic phosphate, as well as two phosphate analogs, beryllium fluoride and orthovanadate, also inhibited cabbage PLD. Enzyme kinetic studies confirmed that PLD followed Hill kinetics, characteristic for allosteric enzymes, with an apparent Hill coefficient (n(app)) of 3.8, indicating positive cooperativity among multiple substrate-binding sites and suggesting possible functional oligomerization of the enzyme. AlF(4)(-) modification of PLD kinetics was consistent with a competitive mode of enzyme inhibition. It is therefore proposed that AlF(4)(-), and other phosphate analogs, inhibits plant PLD by competing with a substrate phosphate group for a substrate-binding site, thereby preventing the formation of an enzyme-phosphatidyl intermediate. This may be a conserved feature of PLD superfamily enzymes.

This work experimentally confirms the pathway of activation of Ha-ras-p21, which was calculated by the method of Targeted Molecular Dynamics (TMD) (Díaz JF, Wroblowski B, Schlitter J, Engelborghs Y, 1997a, Proteins Struct Funct Genet 28:434-451). The process can be studied experimentally by analyzing the binding of BeF3- to the GDP complex of the active fluorescent mutant Y32W (Díaz JF, Sillen A, Engelborghs Y, 1997b, J Biol Chem 227:23138-23143). Two mutants, V29G and 136G, have been constructed at both sides of the effector loop of the active fluorescent mutant. This was done to check the proposed reaction pathway and to provide further insight into the mechanism of the activation of ras proteins. Both mutations accelerate the conformational isomerization with two orders of magnitude, demonstrating convincingly the role of these residues as hinges of the effector loop in one or more of the transitions of the conformational change. These results provide experimental support to the pathway calculated by TMD analysis.

Incubation of the MoFe protein (Kp1) and Fe protein (Kp2), the component proteins of Klebsiella pneumoniae nitrogenase, with BeF(3)(-) and MgADP resulted in a progressive inhibition of nitrogenase activity. We have shown that at high Kp2 to Kp1 molar ratios this inhibition is due to the formation of an inactive complex with a stoichiometry corresponding to Kp1.{Kp2.(MgADP.BeFx)2}2. At lower Kp2:Kp1 ratios, an equilibrium between this 2:1 complex, the partially active 1:1 Kp1.Kp2.(MgADP. BeFx)2 complex, and active nitrogenase components was demonstrated. The inhibition was reversible since incubation of the 1:1 complex in the absence of MgADP and beryllium resulted in complete restoration of activity over 30 h. Under pseudo-first-order conditions with regard to nitrogenase components and MgADP, the kinetics of the rate of inhibition with increasing concentrations of BeF(3)(-) showed a square dependence on [BeF(3)(-)], consistent with the binding of two Be atoms by Kp2 in the complex. Analytical fplc gel filtration profiles of Kp1.Kp2 incubation mixtures at equilibrium resolved the 2:1 complex and the 1:1 complex from free Kp1. Deconvolution of the equilibrium profiles gave concentrations of the components allowing constants for their formation of 2.1 x 10(6) and 5.6 x 10(5) M(-1) to be calculated for the 1:1 and 2:1 complexes, respectively. When the active site concentration of the different species was taken into account, values for the two constants were the same, indicating the two binding sites for Kp2 are the same for Kp1 with one or both sites unoccupied. The value for K(1) we obtain from this study is comparable with the value derived from pre-steady-state studies of nitrogenase. Analysis of the elution profile obtained on gel filtration of a 1:1 ratio incubation mixture containing 20 microM nitrogenase components showed 97% of the Kp2 present initially to be complexed. These data provide the first unequivocal demonstration that Fe protein preparations which may contain up to 50% of a species of Fe protein defective in electron transfer is nevertheless fully competent in complex formation with MoFe protein.

Hitherto ras-related GTP-binding proteins have been considered not to bind phosphate analogs (Kahn, R. A. (1991) J. Biol. Chem. 266, 15595-15597), at least in the absence of activating proteins (Mittal, R., Reza, M., Goody, R., and Wittinghofer, A. (1996) Science 273, 115-117). In this work, we have used a fluorescent active mutant (Y32W) of p21(Ha-)ras to demonstrate that BeF3- binds to the GDP. p21(Ha-ras) complex in the absence of activating proteins. It induces a conformational change leading to a state with fluorescence properties similar to those of the active state. The binding has a low affinity (Kd at 25 degrees C = 8.1 +/- 0.3 mM) and is endothermic (DeltaH = 22.3 +/- 1.6 kJ mol-1). The similarity between the GTP-bound form and the GDP.BeF3--bound form has been confirmed using lifetime analysis of the tryptophan fluorescence. The kinetic analysis of the process indicates that the binding can be divided into a first bimolecular step, which accounts for the association of the anion with its binding site, and a second step, which corresponds to an internal conformational transition of the GDP. BeF3-.p21(Ha-)ras complex to its final state. Both steps are endothermic (DeltaH1 = 15 +/- 2 kJ mol-1 and DeltaH2 = 8 +/- 2 kJ mol-1). The kinetically determined enthalpy change of 23 +/- 4 kJ mol-1 is in excellent agreement with the equilibrium analysis.

Holmes proposed that in F-actin, hydrophobic residues in a subdomain 3/4 loop interact with a hydrophobic pocket on the opposing strand resulting in helix stabilization. We have determined how a decreased hydrophobicity of this plug affects yeast actin function. Cells harboring only the V266G, V266D, V266F, L267G, L269D, or L269K actins appear normal, although V266G cells display an altered budding pattern. However, V266G,L267G (GG) double mutant cells are cold-sensitive with randomly oriented thick actin assemblies seen in rhodamine phalloidin-stained GG cells. V266D actin polymerizes slower than wild-type actin at room temperature. At 4 degrees C, not only is polymerization slowed, but there is also an effect on critical concentration. However, the polymerization defects are milder than those associated with substitution of Asp for the neighboring Leu267. Purified GG-actin does not polymerize in vitro alone or in the presence of wild-type F-actin seeds. GG-actin polymerization can be restored by larger amounts of wild-type actin, beryllium fluoride, or phalloidin at room temperature, although at 4 degrees C only phalloidin is effective. These results suggest that the diminished hydrophobicity of the plug in GG-actin leads to filament destabilization. However, the V266D actin results require a modification of the original Holmes filament model.

The effects of various modifications of rabbit skeletal myosin subfragment 1 on the thermal denaturation of subfragment 1 in ternary complexes with Mg-ADP and orthovanadate (V1) or beryllium fluoride (BeFx) have been studied by differential scanning calorimetry. It has been shown that specific modifications of SH1 group of Cys-707 by different sulfhydryl reagents, trinitrophenylation of Lys-83, and reductive methylation of lysine residues promote the decomposition of the S1.ADP.Vi complex and change the character of structural transitions of the subfragment 1 molecule induced by the formation of this complex, but they have much less or no influence on subfragment 1 thermal stability in the S1.ADP.BeFx complex. Thus, the differential scanning calorimetric studies on modified subfragment 1 preparations reveal a significant difference between S1.ADP.Vi and S1.ADP.BeFx complexes. It is suggested that S1.ADP.Vi and S1.ADP.BeFx complexes represent structural analogues of different transition states of the ATPase cycle, namely the intermediate states S1**.ADP.Pi and S1*.ATP, respectively. It is also proposed that during formation of the S1.ADP.Vi complex the region containing both Cys-707 and Lys-83 plays an important role in the spread of conformational changes from the active site of subfragment 1 ATPase throughout the structure of the entire subfragment 1 molecule. In such a case, the effects of reductive methylation of lysine residues on the subfragment 1 structure in the S1.ADP.Vi complex are related to the modification of Lys-83.

It has been recently shown by differential scanning calorimetry (DSC) that the formation of stable complexes of myosin subfragment 1 (S1) with Mg-ADP and orthovanadate (Vi) or beryllium fluoride (BeFx) causes a global conformational change in the S1 molecule which is reflected in a pronounced increase of S1 thermal stability and in a significant change of S1 domain structure [Shriver, J. W., & Kamath U. (1990) Biochemistry 29, 2556-2564; Levitsky, D. I., Shnyrov, V. L., Khvorov, N. V., Bukatina, A. E., Vedenkina, N. S., Permyakov, E. A., Nikolaeva, O. P., & Poglazov, B. F. (1992) Eur. J. Biochem. 209, 829-835; Bobkov, A. A., Khvorov, N. V., Golitsina, N. L., & Levitsky, D. I. (1993) FEBS Lett. 332, 64-66]. In this work, which continues the previous investigations, we report on a DSC study of the complexes of S1 with various nucleoside diphosphates (NDP). In the absence of Vi or BeFx the various Mg(2+)-NDP and Mg(2+)-PPi had a similar effect on the S1 conformation. All of them had practically no influence on the temperature of the thermal transition but increased its sharpness. However, in the presence of Vi or BeFx the effects of Mg(2+)-NDP complexes were quite different from each other and strongly depended on the base structure of NDP; their effectiveness in inducing conformational changes in S1 and the stability of these complexes decreased in the following order: ADP > CDP >> UDP >> IDP > GDP.(ABSTRACT TRUNCATED AT 250 WORDS)

Fluoroaluminate and fluoroberyllate are potent inhibitors of the ATPase activity of myosin. Inhibition requires the presence of ADP, and much evidence has accumulated to suggest that the tetrahedral fluoroaluminate and fluoroberyllate ions act as phosphate analogues, binding with high affinity at the active site in the position normally occupied by the terminal phosphate of ATP. Both the S1-ADP-fluoroaluminate and the S1-ADP-fluoroberyllate species are thought to resemble kinetic intermediates in the actomyosin ATPase cycle. Characterization of S1-bound fluoroaluminate by 19F NMR is straightforward; a single resonance identified as AlF4- is observed easily [Maruta, S., Henry, G.D., Sykes, B.D., & Ikebe, M (1993) J. Biol. Chem. 268, 7093-7100]. Bound fluoroberyllate, by contrast, was found to give rise to four separate peaks: a downfield pair at -80 and -83.5 ppm and an upfield pair at -101.5 and -103 ppm, suggesting the existence of four distinct types of S1-ADP-fluoroberyllate complex. The relative intensities of the bound resonances can be altered by changing rhe F:Be ratio during complex formation. Integration of a spectrum acquired in the presence of a fluorine-labeled nucleotide derivative, 3'(2')-O-(4-fluorobenzoyl)-ADP, in place of ADP yielded a bound fluoride to nucleotide ratio of 1.7-1.9 to 1, showing that the major bound fluoroberyllate species cannot be BeF3- as is usually thought. It is proposed that the bound fluoroberyllates correspond to the neutral species BeF2(H2O)2 and BeFOH(H2O)2 and the negatively charged species [BeF2OH.H2O]- and [BeF3.H2O]-, although other possibilities are discussed.

The binding of phosphate analogs to F-ADP-actin filaments and their effect on the dynamics of the polymer have been investigated. Orthovanadate binds to F-actin with the same affinity as phosphate and, at low saturation levels, stabilizes the filament structure in a Pi-like fashion; at higher concentration, it promotes destabilization of the filament. BeF-3 and A1F-4 bind to F-ADP-actin in competition with Pi, with a stoichiometry of 1 mol/mol of F-actin subunit and an affinity 3 orders of magnitude higher than Pi (KD = 2 and 25 microM for BeF-3 and A1F-4, respectively). BeF-3 and A1F-4 mimic Pi in stabilizing F-actin and slow down the rate of actin dissociation from filaments 150-fold. Only 1% of F-ADP-BeF3 subunits provide extensive stabilization of the filament. A quantitative analysis of the stabilization by BeF-3 is proposed. While Pi appears in rapid equilibrium with F-ADP-actin, BeF-3 binds to and dissociates from F-ADP-actin at very slow rates (k+ = 4 M-1 S-1; k = 8.10(-6) S-1). In addition, although functionally similar to the reconstituted F-ADP-Pi species, F-ADP-BeF3 has a different conformation as indicated by the 17% quenching of pyrenyl fluorescence linked to BeF-3 binding. We suggest that BeF-3 may be a good analog of the transition state F-ADP-P* and that Pi release following cleavage of ATP on F-actin might be rate-limited by the isomerization of F-ADP-P*.