Study of conformations of the adenosine phosphates. I: Adenosine monophosphate (AMP)

Theoretical studies of the AMP molecule are made in a free and isolated environment with extended Mickel theory (EHT) using the experimentally observed bond lengths and angles, and the experimentally observed torsion angles as a starting conformation. Four torsional degrees of freedom were assumed and all AMP atoms were included in the calculations. Results show that the AMP structure corresponds to an EHT minimum energy configuration when the molecule is isolated from the crystal atoms. This is in contrast with results reported for ATP in the succeeding paper and is attributed to the fact that AMP is crystallized as a free acid. A global minimum, corresponding to a more open structure, is calculated to be more stable than the crystalline form and is lower in energy by 0·05 eV.     

Molecular orbital studies on the conformation of phospholipids. II. Preferred conformations of hydrocarbon chains and molecular organization in biomembranes.

The preferred conformations of the nonpolar β and γ (hydrocarbon) chains in phospholipids have been derived using EHT and CNDO calculations. These calculations indicate that the possible conformations of phospholipids are highly restricted. The calculations find support from X-ray diffraction studies and NMR measurements on model compounds. When considering conformations relevant to structures in cell membranes, a further selection is possible because of the fact that in aqueous solutions hydrophobic interactions stabilize an arrangement where the hydrocarbon chains (β and γ) are stacked almost parallel to one another, leading to a bilayer structure. The various models for β and γ-chains which satisfy this condition have been compared and it has been shown that of these only four are favoured by energy considerations. These arrangements differ from one another in the orientation of the β-chain and γ-chains in the interior of the bilayer structure. A low energy pathway connects these conformations and thus the molecule can easily flip from one stable bilayer arrangement to another. The possible conformations of the polar group (α) are likewise restricted. The proposed model provides explanations to a number of dynamic and static properties of phospholipids, in particular to the observed NMR coupling constants, ¹H and ¹³C relaxation times, studies based on ESR spin labels and the observed X-ray diffraction results on model compounds.     

Calculated minimum energy conformations of nonactin

Nonactin is a cyclic actinomycete metabolite which has been implicated as an ion carrier in the passive transport of potassium across certain biological membranes. In order to discover the conformations of the molecule which are involved in its biochemical function, computer calculations were initiated to derive the energetically favored conformations of the nonactin ring. By assuming that all the relevant three-dimensional conformations of nonactin have the same symmetry property as that suggested by the chemical structure of the molecule (S₄) it was possible to generate a representative sample of all sterically allowed conformations of nonactin. The energies of these conformations were calculated by taking into account the nonbonded interactions among the 116 atoms of the molecule and the torsional potential energy of the 20 rotatable backbone bonds of the ring. The initial results reported in this paper indicate that even in the absence of potassium ion the nonactin ring folds into the same compact tennis-ball seam-like conformation that was found by an X-ray crystallographic investigation of the nonactin/KNCS complex.     

Conformational analysis of phosphatides: Mapping and minimization of the intramolecular energy

Empirical intramolecular energy calculations were carried out on molecular fragments related to phosphatides in order to find the preferred conformations. The energy was mapped as a function of several pairs of torsional angles in progressively larger molecular fragments, with energy minimization being carried out at each map point with respect to other significant variables. The energy mapping results were used as starting points for energy minimization on diheptanoyl L-α-phosphatidic acid-C, which consisted of the named molecule plus a carbon atom attached to one of the phosphate oxygens. It was found that there are 6 pairs of values for 2 of the torsional angles at the 3-way branch point in the glyceryl group which give sterically acceptable conformations; only 4 of these are compatible with lipid bilayer structure in that they can give a parallel arrangement of the acyl chains. The several acceptable conformations of the phosphate and acyl ester groups within each of these conformational classes are enumerated. The results obtained may be used as a guide for further experimental and theoretical work on phosphatide structures.     

Inhibition of recA protein promoted ATP hydrolysis. 1. ATP gamma S and ADP are antagonistic inhibitors

ADP and adenosine 5'-O-(3-thiotriphosphate) (ATP gamma S) inhibit recA protein promoted ATP hydrolysis by fundamentally different mechanisms. In both cases, at least two modes of inhibition are observed. For ADP, the first mode is competitive inhibition. The second mode is manifested by dissociation of recA protein from DNA. These are readily distinguished in a comparison of ATP hydrolyses that are activated by (a) DNA and (b) high (approximately 2 M) salt concentrations. Competitive inhibition with a significant degree of cooperativity is observed under both sets of conditions, although the DNA-dependent activity is more sensitive to ADP than the high-salt reaction. The reaction in the presence of poly(deoxythymidylic acid) or duplex DNA ceases when about 60% of the available ATP is hydrolyzed, reflecting an ADP-mediated dissociation of recA protein from the DNA that is governed by the ADP/ATP ratio. In contrast, ATP hydrolysis proceeds nearly to completion at high salt concentrations. At high concentrations of ATP and ATP gamma S, ATP gamma S also acts as a competitive inhibitor. At low concentrations of ATP gamma S and ATP, however, ATP gamma S activates ATP hydrolysis. These patterns are observed for recA-mediated ATP hydrolysis with either high salt concentrations or a poly(deoxythymidylic acid) [poly(dT)] cofactor, although the activation is observed at much lower ATP and ATP gamma S concentrations when poly(dT) is used. ATP gamma S can also relieve the inhibitory effect of ADP under some conditions. ATP gamma S and ADP are antagonistic inhibitors, reinforcing the idea that they stabilize different conformations of the protein and suggesting that these conformations are mutually exclusive. The ATP gamma S (ATP) conformation is active in ATP hydrolysis. The ADP conformation is inactive.     

The conformational analysis of adenosine triphosphate by classical potential energy calculations

The conformational analysis of adenosine triphosphate was conducted by using classical potential energy calculations. All rotatable bonds were examined, i.e., no dihedral angles were fixed at predetermined conformations except for the ribofuranose ring, which was held in the C(3′)-endo conformation—the conformation observed for adenosine in the crystal state. The energy terms included in the total energy expression consist of nonbonded pairwise interaction, electrostatic pairwise interaction, free energy of solvation, and torsional bond potentials. Two separate approaches were used in the conformational analyses. The first consisted of a sequential fragment approach were four bonds were rotated simultaneously at 30° increments. Each fragment overlapped the preceding one by at least one bond. All rotors were then simultaneously examined at their minima and at ±15°. The second approach consisted of a coarse grid search where all rotors were examined simultaneously, but only at staggered positions. The low-energy conformations thus obtained were then used as starting conformations for a minimization routine based on the method of conjugate directions. The first approach required about 40 hr of central processing unit (CPU) computer time, while the coarse grid/minimization approach required about 4 hr of CPU time. Both the sequential fragment approach and the minimization approach yielded lowest-energy conformations which are remarkably similar to the solid-state conformation of C(3′)-endo ATP.     

Conformations of carnitine and acetylcarnitine and the relationship to mitochondrial transport of fatty acids

The conformations of carnitine and acetylcarnitine by EHT and CNDO/2 molecular orbital calculations show that carnitine has two low energy conformers. One of these is an extended conformer corresponding to a charge separated species, while the other is a folded conformer having a charged onium head and carboxylate anion in close proximity forming an internal ionic bond. These results suggest that the folded conformation is responsible for the active transport of acetyl- and acyl-carnitine by enzymes which transfer acyl groups into the mitochondria for subsequent fatty acid oxidation.     

Structural characterization of manganese(II)-nucleotide complexes bound to yeast 3-phosphoglycerate kinase: 13C relaxation measurements using [U-13C]ATP and [U-13C]ADP

A complete characterization of the conformations of Mn.ADP and Mn.ATP bound to the active site of yeast 3-P-glycerate kinase is presented. These conformations have been deduced on the basis of paramagnetic effects on 13C spin-lattice relaxation rates in [U-13C]nucleotides due to Mn(II), used as a substituent activating cation. The 13C relaxation measurements were performed on exclusively enzyme-bound complexes E.Mn.[U-13C]ATP and E.Mn.[U-13C]ADP at three distinct 13C NMR frequencies: 75.4, 125.7, and 181 MHz. The frequency dependence of the relaxation data has been analyzed in an effort to evaluate distances from the cation for all 10 13C nuclei in the adenosine moieties of E.Mn.ATP and E.Mn.ADP. These distance data, taken along with previously published cation-31P distances, have been used as constraints in the molecular modeling program Quanta, in which molecular dynamics simulations and energy minimization have been performed to determine the conformations that are compatible with the distance data. It was possible to model the distances on the basis of a single enzyme-bound conformation for each of the nucleotides. The details of the enzyme-bound Mn.ATP and Mn.ADP conformations are distinguishably different from each other, indicating that structural alterations occur in the enzyme-bound reaction complex as the enzyme turns over. For example, when the adenosine moieties in the bound structures of Mn.ATP and Mn.ADP are superposed, the cation is found to be displaced by approximately 2.4 A between the two conformations, suggesting that these structural changes may involve movements with significant amplitudes. Furthermore, the NMR-determined structures of enzyme-bound Mn.ATP and Mn.ADP are significantly different from those in published X-ray crystal structures of the enzyme-nucleotide complexes.     

Inhibition of recA protein promoted ATP hydrolysis. 2. Longitudinal assembly and disassembly of recA protein filaments mediated by ATP and ADP

There are at least two major conformations of recA nucleoprotein filaments formed on poly-(deoxythymidylic acid) [poly(dT)], one stabilized by ATP [or adenosine 5'-O-(3-thiotriphosphate) (ATP gamma S)] and one stabilized by ADP. Assembly of filaments in the ATP conformation is much faster than assembly in the ADP conformation. A third conformation may be present in the absence of nucleotides. The ATP and ADP conformations are mutually exclusive. When a mixture of ATP and ADP is present, recA protein binding is a function of the ADP/ATP ratio. Complete dissociation is observed when the ratio becomes 1.0-1.5. When a mixture of ATP and ADP is present at the beginning of a reaction, a transient phase lasting several minutes is observed in which the system approaches the state characteristic of the new ADP/ATP ratio. This phase is manifested by a lag in ATP hydrolysis when ATP is added to preformed ADP filaments, and by a burst in ATP hydrolysis in all other cases. More than 15 ATPs are hydrolyzed per bound recA monomer during the burst phase. The transient phase reflects an end-dependent disassembly process propagated longitudinally through the filament, rather than a slow conformation change in individual recA monomers or a slow exchange of one nucleotide for the other. The hysteresis exhibited by the system provides a number of insights relevant to the mechanism of recA-mediated DNA strand exchange.     

Chloroplast membranes and coupling factor conformations.

The demonstrated role of proton translocation and resulting electrochemical activity gradients (protonmotive force) in ATP synthesis by chloroplasts is noted. Evidence for the participation of conformational changes in the terminal ATPase (coupling factor, or CF1) is reviewed. Hydrogen exchange into ordinarily cyptic groups of the molecule occurs only when the subtending membranes are put under the stress of a protonmotive force. Since up to 100 hydrogen atoms per mole are involved in the energy-dependent exchange the conformational change permitting tham access to the medium must be a major one. Chemical reagents are beginning to be used to attack groups on CF1 that are exposed only when the membranes are energized. N-ethylmaleimide binds covalently, sulfate causes as yet unspecified damage, and permanganate leads to oxidative damage to CF1 under energized conditions. The last two reagents are analogues of phosphate, and ADP must be added for them to inhibit. On the basis of this and other differences between the conditions needed for inhibition by permanganate or sulfate, and that by N-ethylmaleimide or the hydrogen exchange, a somewhat complex scheme involving several successive or alternative conformations of CF1 can be postulated. Questions are raised as to the way in which a conformational change in a bound protein could be caused by a proton activity gradient across its supporting membrane, and as to whether the altered conformations might constitute a part of the energy transformations leading to ATP synthesis.     

Understanding ATP synthesis: structure and mechanism of the F1-ATPase (Review)

To couple the energy present in the electrochemical proton gradient, established across the mitochondrial membrane by the respiratory chain, to the formation of ATP from ADP and Pi, ATP-synthase goes through a sequence of coordinated conformational changes of its major subunits (alpha, beta). These changes are induced by the rotation of the gamma subunit driven by the translocation of protons through the c subunit of the membrane portion of the enzyme. During this process, the F1-portion of the ATP-synthase adopts at least two major conformations depending on the occupancy of the beta subunits: one with two nucleotides, the other with three. In the two-nucleotide structure, the empty beta subunit adopts an open conformation that is highly different from the other conformations of beta subunits: tight, loose and closed. The three-dimensional structures of the F1-ATPase in each of these two major conformations provide a framework for understanding the mechanism of energy coupling by the enzyme. The energetics associated with two different models of the reaction steps, analysed using molecular dynamics calculations, show that three-nucleotide intermediates do not occur in configurations with an open beta subunit; instead, they are stabilized by completing a jaw-like motion that closes the beta subunit around the nucleotide. Consequently, the energy driven, major conformational change takes place with the beta subunits in the tight, loose and closed conformation.     

Binding of nucleotides to nucleoside diphosphate kinase monitored by rose Bengal

The binding of nucleotides to nucleoside-diphosphate kinase from pig heart was studied using the dye rose Bengal as an optical probe. By difference absorption spectroscopy and by equilibrium dialysis it was shown that one dye molecule strongly bound per enzyme subunit. By competition with nucleotides it was shown that two nucleotide-binding sites exist on each subunit of either unphosphorylated or phosphorylated enzyme: one of them binds ATP or ADP tightly, the other one binds rose Bengal tightly and ADP loosely. As detected by different affinities for rose Bengal the enzyme exists in two conformations: a 'relaxed' conformation induced by the binding of ADP, and a 'tense' conformation induced by the binding of ATP or by phosphorylation.     

Insights into the chemomechanical coupling of the myosin motor from simulation of its ATP hydrolysis mechanism

The molecular motor myosin converts chemical energy from ATP hydrolysis into mechanical work, thus driving a variety of essential motility processes. Although myosin function has been studied extensively, the catalytic mechanism of ATP hydrolysis and its chemomechanical coupling to the motor cycle are not completely understood. Here, the catalysis mechanism in myosin II is examined using quantum mechanical/molecular mechanical reaction path calculations. The resulting reaction pathways, found in the catalytically competent closed/closed conformation of the Switch-1/Switch-2 loops of myosin, are all associative with a pentavalent bipyramidal oxyphosphorane transition state but can vary in the activation mechanism of the attacking water molecule and in the way the hydrogens are transferred between the heavy atoms. The coordination bond between the Mg2+ metal cofactor and Ser237 in the Switch-1 loop is broken in the product state, thereby facilitating the opening of the Switch-1 loop after hydrolysis is completed, which is required for subsequent strong rebinding to actin. This reveals a key element of the chemomechanical coupling that underlies the motor cycle, namely, the modulation of actin unbinding or binding in response to the ATP or ADP x P(i) state of nucleotide-bound myosin.     

Conformation and hydration of aspartame

Conformational free energy calculations using an empirical potential (ECEPP/2) and the hydration shell model were carried out on the neutral, acidic, zwitterionic, and basic forms of aspartame in the hydrated state. The results indicate that as the molecule becomes more charged, the number of low energy conformations becomes smaller and the molecule becomes less flexible. The calculated free energies of hydration of charged aspartames show that hydration has a significant effect on the conformation in solution. Only two feasible conformations were found for the zwitterionic form, and these are consistent with the conformations deduced from NMR and X-ray diffraction experiments. The calculated free energy difference between these two conformations was 1.25 kcal/mol. The less favored of the two solvated conformations can be expected to be stabilized by hydrophobic interaction of the phenyl groups in the crystal.     

Nuclear overhauser effect studies of the conformations of Mg(alpha, beta-methylene)ATP bound to E. coli isoleucyl-tRNA synthetase

Internuclear distances obtained from transferred nuclear Overhauser effects were used in combination with distance geometry calculations to define the E. coli isoleucyl-tRNA synthetase bound conformation of Mg(alpha, beta-methylene)ATP both in the absence and in the presence of the cognate and noncognate amino acids L-isoleucine and L-valine, respectively. A single nucleotide structure having an anti adenine-ribose glycosidic torsional angle of -114 degrees was found to satisfy the experimental distance constraints. The nearly identical anti glycosidic torsional angles observed in all three complexes demonstrate that the conformation of the adenosine moiety of the enzyme-bound nucleotide is not sensitive to the presence or to the nature of the amino acid bound at the aminoacyladenylate site. In addition, the acceptable range of Mg(alpha, beta-methylene)ATP conformations bound to the E. coli isoleucyl-tRNA synthetase was found to be nearly identical to that previously determined for the E. coli methionyl-tRNA synthetase (Williams and Rosevear (1991) J. Biol. Chem. 266, 2089-2098). Thus, the predicted structural homology between the isoleucyl- and methionyl-tRNA synthetases, both members of the same class of synthetases on the basis of common consensus sequences, is further supported by consensus enzyme-bound nucleotide conformations.     

Stereochemistry of the interaction between methionine sulfur and the protein core.

The stereochemical features of the interaction between the sulfur atom of methionine residues and surrounding atoms are examined on a large set of known protein crystal structures. It appears that the minimum energy conformations observed in small molecule crystals are not observed within the protein core. This suggests that these interactions are either of little intensity, though they might contribute to regulate the protein physiological behavior, or physicochemically different from their counterpart in small molecule crystals.     

Projection structure of the atractyloside-inhibited mitochondrial ADP/ATP carrier of Saccharomyces cerevisiae

ADP/ATP carriers in the inner mitochondrial membrane catalyze the exchange of cytosolic ADP for ATP synthesized in the mitochondrial matrix by ATP synthase and thereby replenish the eukaryotic cell with metabolic energy. The yeast ADP/ATP carrier (AAC3) was overexpressed, inhibited by atractyloside, purified, and reconstituted into two-dimensional crystals. Images of frozen hydrated crystals were recorded by electron microscopy, and a projection structure was calculated to 8-A resolution. The AAC3 molecule has pseudo 3-fold symmetry in agreement with the 3-fold sequence repeats that are typical of members of the mitochondrial carrier family. The density distribution is consistent with a bundle of six transmembrane alpha-helices with two or three short alpha-helical extensions closing the central pore on the matrix side. The AAC3 molecules in the crystal are arranged in symmetrical homo-dimers, but the translocation pore for adenine nucleotides lies in the center of the molecule and not along the dyad axis of the dimer.     

Preferred phosphodiester conformations in nucleic acids. A virtual bond torsion potential to estimate lone-pair interactions in a phosphodiester

The lone-pair orbital interactions arising in a phosphodiester are incorporated into semiempirical conformational energy calculations using a unifold “torsional potential” around the virtual bond linking the ester oxygen atoms. The results explain the observed experimental data better than other methods.     

Quantum chemical studies on the conformational structure of nucleic acids. IV. Calculation of backbone structure by CNDO method

Quantum chemical calculations using the CNDO/2 method, have been carried out to determine the energetically favoured ranges of the torsional angles (φ′, ω′, ω, φ, ψ) which fix the conformational structure of nucleic acid backbone. The two dimensional isoenergy maps have been constructed in the (ω′, ω) and (φ, ψ) hyperspaces. The variation of total energy with respect to φ′ has also been studied. The results show that the non-bonding interactions play a major role in the conformational stability of nucleic acids and polynucleotides. The theoretical predictions show good correspondence with the experimental data (X-ray and ¹³C NMR) as well as the other reported theoretical calculations (EHT, PCILO and classical potential functions). The most favoured structure has the conformational angles close to 240, 290, 290, 180 and 60° and these values lead to a helical structure with a pitch of 34 Å and about ten nucleotide units per turn of the helix. The proposed models of Watson &; Crick, DNA-B and DNA-C lie in high energy regions.     

The structure of bovine F1-ATPase inhibited by ADP and beryllium fluoride

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.