General features in the stoichiometry and stability of ionophore A23187-cation complexes in homogeneous solution

Existing literature describing the stoichiometry and stability of complexes between A23187 and divalent cations in solution has been extended to include additional transition series cations, the heavy-metal cations Cd2+ and Pb2+, plus seven lanthanide series trivalent cations. Stability constants of 1:1 complexes between the ionophore and the divalent cations vary by 6.2 orders of magnitude between Cu2+ and Ba2+ which are the strongest and weakest complexes, respectively. Considering alkaline-earth and first-series transition cations together, the pattern of stability constants obeys the extended Irving-Williams series as is seen with many nonionophorous liganding agents. Cd2+ and Pb2+ are bound with an affinity similar to those of Mn2+ and Zn2+, whereas the lanthanides are bound with little selectivity and slightly higher stability. Titration of the ionophore in the 10(-5) M concentration range with di- and trivalent cations gives rise first to complexes of stoichiometry MA2 and subsequently to MA as the metal concentration is increased. The second stepwise stability constants for formation of the MA2 species exceeds the first constant by approximately 10-fold. With lanthanides, heavy metals, and transition-metal cations, OH-, at near physiological concentrations, competes significantly with free ionophore for binding to the 1:1 complexes. This competition is not apparent when Ca2+ or Mg2+ are the central cations. Possible implications of the 1:1 complex selectivity pattern, the ionophore-hydroxide competitive binding equilibria, and potential ternary complexes involving 1:1 ionophore:cation complexes and other anions present in biological systems are discussed with respect to the ionophore's transport selectivity and biological actions.     

Transition metal complexes of terminally protected peptides containing histidyl residues

Histidine-containing peptide fragments of prion protein are efficient ligands to bind various transition metal ions and they have high selectivity in metal binding. The metal ion affinity follows the order: Pd(II)>Cu(II)>Ni(II)Zn(II)>Cd(II) approximately Co(II)>Mn(II). The high selectivity of metal binding is connected to the involvement of both imidazole and amide nitrogen atoms in metal binding for Pd(II), Cu(II) and Ni(II), while only the monodentate N(im)-coordination is possible with the other metal ions. The stoichiometry and binding mode of palladium(II) complexes show great variety depending on the metal ion to ligand ratio, pH and especially the presence of coordinating donor atoms in the side chains of peptide fragments. It is also clear from our data that the peptide fragments containing histidine outside the octarepeat (His96, His111 and His187) are more efficient ligands than the monomer peptide fragments of the octarepeat domain.     

Genetic competence in Escherichia coli requires poly-beta-hydroxybutyrate/calcium polyphosphate membrane complexes and certain divalent cations.

In earlier studies of genetic competence in Escherichia coli induced with calcium-containing buffers, a strong correlation was found between transformation efficiency and the formation of poly-beta-hydroxybutyrate/calcium polyphosphate (PHB/Ca2+/PPi) complexes in the plasma membranes. In this study, we replaced Ca2+ with one of a number of other cations--monovalent, divalent, and trivalent--and found significant numbers of transformants (transformation efficiency, > 10(5)/micrograms of pBR322 DNA) only when the cells had high levels of PHB/Ca2+/PPi and the medium contained at least one of the divalent cations Ca2+, Mn2+, Sr2+, or Mg2+. Cells with high levels of the complexes were not competent when the medium did not contain these cations, but the cations were also ineffectual when the cells had few complexes. Surprisingly, Mn, Sr, and Mg were not incorporated into the complexes in place of Ca. These results indicate that PHB/Ca2+/PPi complexes and the above-mentioned divalent cations each have essential but disparate roles in genetic competence. Moreover, the strong selectivity of PHB/PPi for Ca2+ suggests the binding sites in the complexes are ionophoretic.     

Design, synthesis, and evaluation of peptidyl fluorescent probe for Zn2+ in aqueous solution

A new fluorescent peptide probe for the detection of Zn(2+) was synthesized on the basis of zinc binding ligands in zinc enzymes. The peptide that has a unique amino acid sequence displayed a great selectivity for Zn(2+) in the presence of several transition metal ions in aqueous solution. The reversibility, binding stoichiometry, binding affinity, and pH sensitivity of the sensor were studied. Further, on-bead application of the peptide as chemosensors was demonstrated.     

Conservative and nonconservative mutations of the transmembrane CPC motif in ZntA: effect on metal selectivity and activity

ZntA from Escherichia coli belongs to the P1B-ATPase transporter family and mediates resistance to toxic levels of selected divalent metal ions. P1B-type ATPases can be divided into subgroups based on substrate cation selectivity. ZntA has the highest selectivity for Pb2+, followed by Zn2+ and Cd2+; it also shows low levels of activity with Cu2+, Ni2+, and Co2+. It has two high-affinity metal-binding sites, one each in the N-terminus and the transmembrane domains. Ligands to the transmembrane metal site in ZntA include the cysteine residues of the conserved 392CPC394 motif in the sixth transmembrane helix. Pro393 is invariant in all P-type ATPases. For ZntA homologues with different metal ion selectivity, the cysteines are replaced by serine, histidine, and threonine. To test the effect on activity and metal ion selectivity, single alanine, histidine, and serine substitutions at Cys392 or Cys394 in ZntA were characterized, as well as double substitutions of both cysteines by histidine or serine. P393A was also characterized. C392A, C394A, and P393A lost the ability to bind a metal ion with high affinity in the transmembrane domain. Histidine and serine substitutions at Cys392 and Cys394 resulted in loss of binding of Pb2+ at the transmembrane site, indicating that both cysteines of the CPC motif are required for binding Pb2+ with high affinity in ZntA homologues. However, C392H, C392S, C394H, C394S, C392S/C394S, and C392H/C394H could bind other divalent metal ions at the transmembrane site and retained low but measurable activity. Interestingly, these mutants lost the predominant selectivity for Zn2+ and Cd2+ shown by wtZntA. Therefore, conserved residues contribute to metal selectivity by supplying ligands that bind metal ions not only with high affinity, as for Pb2+, but also with the most favorable binding geometry that results in efficient catalysis.     

Ionophore A23187: the effect of H+ concentration on complex formation with divalent and monovalent cations and the demonstration of K+ transport in mitochondria mediated by A23187.

The two-phase extraction technique has been used to study the equilibrium between A23187, metal cations, and H+. Under these conditions the ionophore forms charge neutral isostoichiometric complexes with divalent cations in which both carboxylate groups of the 2:1 A23187:M2+ complexes are deprotonated. In ethanol, however, the methyl ester of A23187 also binds divalent cations indicating that protonated complexes between A23187 and cations should also exist. With monovalent cations, A23187 forms two charge-neutral complexes of stoichiometries and relative stabilities: A2HM greater than AM. Examination of energy utilization K+ and H+ movements, and light scattering capacity of mitochondria in the presence of divalent cation chelators, A23187, and valinomycin demonstrates that A23187 can act as a nigericin type K+ ionophore under appropriate conditions. Formation constants for the A2HM complexes with monovalent cations indicate that with appropriate conditions transport of Li+ and Na+ mediated by A23187 would also be expected. The binding constant data and associated free energies of complex formation are compared as a function of ionic radius and of cation charge. The data indicate that lack of conformational mobility in A23187 is responsible for the high cation size selectivity of this compound. To explain the transport selectivity of A23187 for divalent cations, it is proposed that this ionophore forms a family of five complexes, isostoichiometric between cations of different valence but of which only charge-neutral species are permeant to membranes. The charge of a given complex is in turn determined by that of the cation. The concept is consistent with the divalent cation transport specificity of A23187, explains the observed monovalent cation transport, and is useful in rationalizing the differences in charge selectivity between A23187 and X-537A.     

Spectroscopic, mass spectrometry, and semiempirical investigation of a new ester of Monensin A with ethylene glycol and its complexes with monovalent metal cations

A new ester of Monensin A with ethylene glycol (MON2) has been synthesized by a new method and its ability to form complexes with Li+, Na+, and K+ cations has been studied by ESI MS, 1H and 13C NMR, FT-IR, and PM5 semiempirical methods. It is demonstrated that MON2 forms stable complexes of 1:1 stoichiometry with monovalent metal cations. The structures of the complexes are stabilized by intramolecular hydrogen bonds in which the OH groups are always involved. In the structure of MON2 the oxygen atom of the C=O ester group is involved in very weak bifurcated intramolecular hydrogen bonds with two hydroxyl groups, whereas in the complexes of MON2 with monovalent metal cations the C=O ester group is not engaged in any intramolecular hydrogen bonds. The structures of the MON2 and its complexes with Li+, Na+, and K+ cations are visualized and discussed in detail.     

Crystallographic comparison of manganese- and iron-dependent homoprotocatechuate 2,3-dioxygenases

The X-ray crystal structures of homoprotocatechuate 2,3-dioxygenases isolated from Arthrobacter globiformis and Brevibacterium fuscum have been determined to high resolution. These enzymes exhibit 83% sequence identity, yet their activities depend on different transition metals, Mn2+ and Fe2+, respectively. The structures allow the origins of metal ion selectivity and aspects of the molecular mechanism to be examined in detail. The homotetrameric enzymes belong to the type I family of extradiol dioxygenases (vicinal oxygen chelate superfamily); each monomer has four betaalphabetabetabeta modules forming two structurally homologous N-terminal and C-terminal barrel-shaped domains. The active-site metal is located in the C-terminal barrel and is ligated by two equatorial ligands, H214NE1 and E267OE1; one axial ligand, H155NE1; and two to three water molecules. The first and second coordination spheres of these enzymes are virtually identical (root mean square difference over all atoms, 0.19 A), suggesting that the metal selectivity must be due to changes at a significant distance from the metal and/or changes that occur during folding. The substrate (2,3-dihydroxyphenylacetate [HPCA]) chelates the metal asymmetrically at sites trans to the two imidazole ligands and interacts with a unique, mobile C-terminal loop. The loop closes over the bound substrate, presumably to seal the active site as the oxygen activation process commences. An "open" coordination site trans to E267 is the likely binding site for O2. The geometry of the enzyme-substrate complexes suggests that if a transiently formed metal-superoxide complex attacks the substrate without dissociation from the metal, it must do so at the C-3 position. Second-sphere active-site residues that are positioned to interact with the HPCA and/or bound O2 during catalysis are identified and discussed in the context of current mechanistic hypotheses.     

Synthesis and X-ray crystal structures of two transition metal complexes based on functionalised 1,5-anhydro-2-deoxy-D-galactitol and methyl 2-deoxy-alpha-D-galactopyranoside

Two new ligands of transition metal cations based on galactose-derived scaffolds were synthesised: 1,5-anhydro-2-deoxy-3,4,6-tri-O-(2-picolyl)-D-galactitol and methyl 2-deoxy-3,4,6-tri-O-(2-picolyl)-alpha-D-galactopyranoside. These ligands permitted the isolation as single crystals of a Co(II) and a Ni(II) complex, respectively. The structures of both complexes were determined by X-ray crystallography showing a coordination sphere including sugar-bound oxygen atoms. The sugar-derived ligands were found to be in both cases in high energy conformations in the crystal structures of the complexes. These conformations contain an arrangement of sugar-bound oxygen atoms similar to those observed in polyol-metal and carbohydrate-metal complexes.     

Metal ion probing of rRNAs: evidence for evolutionarily conserved divalent cation binding pockets.

Ribosomes are multifunctional RNP complexes whose catalytic activities absolutely depend on divalent metal ions. It is assumed that structurally and functionally important metal ions are coordinated to highly ordered RNA structures that form metal ion binding pockets. One potent tool to identify the structural surroundings of high-affinity metal ion binding pockets is metal ion-induced cleavage of RNA. Exposure of ribosomes to divalent metal ions, such as Pb2+, Mg2+, Mn2+, and Ca2+, resulted in site-specific cleavage of rRNAs. Sites of strand scission catalyzed by different cations accumulate at distinct positions, indicating the existence of general metal ion binding centers in the highly folded rRNAs in close proximity to the cleavage sites. Two of the most efficient cleavage sites are located in the 5' domain of both 23S and 16S rRNA, regions that are known to self-fold even in the absence of ribosomal proteins. Some of the efficient cleavage sites were mapped to the peptidyl transferase center located in the large ribosomal subunit. Furthermore, one of these cleavages was clearly diminished upon AcPhe-tRNA binding to the P site, but was not affected by uncharged tRNA. This provides evidence for a close physical proximity of a metal ion to the amino acid moiety of charged tRNAs. Interestingly, comparison of the metal ion cleavage pattern of eubacterial 70S with that of human 80S ribosomes showed that certain cleavage sites are evolutionarily highly conserved, thus demonstrating an identical location of a nearby metal ion. This suggests that cations, bound to evolutionarily constrained binding sites, are reasonable candidates for being of structural or functional importance.     

Multinuclear magnetic resonance, electrospray ionization-mass spectroscopy, and parametric method 5 studies of a new derivative of gossypol with 2-thiophenecarbohydrazide as well as its complexes with LI+, Na+, K+, RB+, and Cs+ cations

A new derivative of racemic gossypol with 2-thiophenecarbohydrazide (GHHT) and its complexes with monovalent cations have been synthesized and studied by electrospray ionization-mass spectroscopy (ESI-MS), multinuclear nuclear magnetic resonance (NMR), as well as by the Parametric Method 5 (PM5) methods. It is demonstrated that GHHT forms stable complexes of 1:1 stoichiometry with monovalent metal cations. The structures of the complexes are stabilized by three types of intramolecular hydrogen bonds. The spectroscopic methods have provided clear evidence that GHHT and its complexes exist in the DMSO-d6 solution in the N-imine-N-imine tautomeric forms. The structures of the GHHT and its complexes with Li+, Na+, K+, Rb+, and Cs+ cations are visualized and discussed in detail.     

Role of transition metal ferrocyanides (II) in chemical evolution

Due to ease of formation of cyanide under prebiotic conditions, cyanide ion might have formed stable complexes with transition metal ions on the primitive earth. In the course of chemical evolution insoluble metal cyano complexes, which settled at the bottom of primeval sea could have formed peptide and metal amino acid complexes through adsorption processes of amino acids onto these metal cyano complexes.Adsorption of amino acids such as glycine, aspartic acid, and histidine on copper ferrocyanide and zinc ferrocyanide have been studied over a wide pH range of 3.6 – 8.5. Amino acids were adsorbed on the metal ferrocyanide complexes for different time periods. The progress of the adsorption was followed spectro-photometrically using ninhydrin reagent. Histidine was found to show maximum adsorption on both the adsorbents at neutral pH. Zinc ferrocyanide exhibits good sorption behaviour for all the three amino acids used in these investigations.     

Structural characteristics of protein binding sites for calcium and lanthanide ions

Surveys of X-ray structures of Ca2+-containing and lanthanide ion-containing proteins and coordination complexes have been performed and structural features of the metal binding sites compared. A total of 515 structures of Ca2+-containing proteins were considered, although the final data set contained only 44 structures and 60 Ca2+ binding sites with a total of 323 ligands. Eighteen protein structures containing lanthanide ions were considered with a final data set containing eight structures and 11 metal binding sites. Structural features analysed include coordination numbers of the metal ions, the identity of their ligands, the denticity of carboxylate ligands, and the type of secondary structure from which the ligands are derived. Three general types of calcium binding site were identified in the final data set: class I sites supply the Ca2+ ligands from a continuous short sequence of amino acids; class II sites have one ligand supplied by a part of the amino acid sequence far removed from the main binding sequence; and class III sites are created by amino acids remote from one another in the sequence. The abundant EF-hand type of Ca2+ binding site was under-represented in the data set of structures analysed as far as its biological distribution is concerned, but was adequately represented for the chemical survey undertaken. A turn or loop structure was found to provide the bulk of the ligands to Ca2+, but helix and sheet secondary structures are slightly better providers of bidentate carboxylate ligation than turn or loop structures. The average coordination number for Ca2+ was 6.0, though for EF-hand sites it is 7. The average coordination number of a lanthanide ion in an intrinsic protein Ca2+ site was 7.2, but for the adventitious sites was only 4.4. A survey of the Cambridge Structural Database showed there are small-molecule lanthanide complexes with low coordination numbers but it is likely that water molecules, which do not appear in the electron density maps, are present for some lanthanide sites in proteins. A detailed comparison of the well-defined Ca2+ and lanthanide ion binding sites suggests that a reduction of hydrogen bonding associated with the ligating residues of the binding sites containing lanthanide ions may be a response to the additional positive charge of the lanthanide ion. Major structural differences between Ca2+ binding sites with weak and strong binding affinities were not obvious, a consequence of long-range electrostatic interactions and metal ion-induced protein conformational changes modulating affinities.     

Estimation of pseudopeptide metal ion complexation tendencies by special MS methods,HPCE and circular dichroism

Summary To obtain more insight into catalytic mechanisms of metallo enzymes and specific metal complexation by proteins we use linear and cyclic pseudopeptides as mimetics. Knowledge about tendencies of complex formation of different ligands with selected transition metal ions is an indispensable prerequisite for the development of homo-and hetero-dinuclear metallo enzyme mimetics. Three pseudotripeptide ligands were investigated with respect to formation tendency and properties of complexes with the transition metal ions Cu²⁺, Zn²⁺ Ni²⁺, Co²⁺ and Mn²⁺. To study complexation tendencies we applied different methods. One of the important prerequisites for the application in a secreening of series of peptide ligands is the necessity for a minimal amount of substance. We used and compared certain masspectrometric methods for the estimation of a rank order of complexation of certain transition metal ions. We also applied spectrophotometric titration, circular dichroism measurements, capillary electrophoresis and pH-rate profile of catalytic activity in the attempt to evaluate complex formation tendencies. Except for the spectrophotometric pH-titration and the pH-profile of catalytic activity all methods, were applicable, but each method has its advantages and disadvantages depending on the separation effect of the ligand from the metal complex, and depending on the spectroscopic properties of ligand and complex. The results regarding complex formation are compared to each other. Comparison of pairs by MALDI-TOF-and ESI-MS allows an estimation of the rank order of complexation tendency of one ligand with different metal ions and requires the least amount of substance. The other investigated methods provided additional information on structural properties of the formed complexes; however either they required too much pseudopeptide ligand or were not applicable for all transition metal ions used in this study.     

Novel conantokins from Conus parius venom are specific antagonists of N-methyl-D-aspartate receptors

We report the discovery and characterization of three conantokin peptides from the venom of Conus parius. Each peptide (conantokin-Pr1, -Pr2, and -Pr3) contains 19 amino acids with three gamma-carboxyglutamate (Gla) residues, a post-translationally modified amino acid characteristic of conantokins. The new peptides contain several amino acid residues that differ from previous conantokin consensus sequences. Notably, the new conantokins lack Gla at the 3rd position from the N terminus, where the Gla residue is replaced by either aspartate or by another post-translationally modified residue, 4-trans-hydroxyproline. Conantokin-Pr3 is the first conantokin peptide to have three different post-translational modifications. Conantokins-Pr1 and -Pr2 adopt alpha-helical conformations in the presence of divalent cations (Mg2+ and Ca2+) but are generally unstructured in the absence of divalent cations. Conantokin-Pr3 adopts an alpha-helical conformation even in the absence of divalent cations. Like other conantokins, the new peptides induced sleep in young mice and hyperactivity in older mice upon intracranial injection. Electrophysiological assays confirmed that conantokins-Pr1, -Pr2, and -Pr3 are N-methyl-d-aspartate (NMDA) receptor antagonists, with highest potency for NR2B-containing NMDA receptors. Conantokin-Pr3 demonstrated approximately 10-fold selectivity for NR2B-containing NMDA receptors. However, conantokin-Pr2 showed minimal differences in potency between NR2B and NR2D. Conantokins-Pr1, -Pr2, and -Pr3 all demonstrated high specificity of block for NMDA receptors, when tested against various ligand-gated ion channels. Conus parius conantokins allow for a better definition of structural and functional features of conantokins as ligands targeting NMDA receptors.     

Estimation of pseudopeptide metal ion complexation tendencies by special MS methods, HPCE and circular dichroism

To obtain more insight into catalytic mechanisms of metallo enzymes and specific metal complexation by proteins we use linear and cyclic pseudopeptides as mimetics. Knowledge about tendencies of complex formation of different ligands with selected transition metal ions is an indispensable prerequisite for the development of homo- and hetero-dinuclear metallo enzyme mimetics. Three pseudotripeptide ligands were investigated with respect to formation tendency and properties of complexes with the transition metal ions Cu²⁺, Zn²⁺, Ni²⁺, Co²⁺ and Mn²⁺. To study complexation tendencies we applied different methods. One of the important prerequisites for the application in a screening of series of peptide ligands is the necessity for a minimal amount of substance. We used and compared certain masspectrometric methods for the estimation of a rank order of complexation of certain transition metal ions. We also applied spectrophotometric titration, circular dichroism measurements, capillary electrophoresis and pH-rate profile of catalytic activity in the attempt to evaluate complex formation tendencies. Except for the spectrophotometric pH-titration and the pH-profile of catalytic activity all methods were applicable, but each method has its advantages and disadvantages depending on the separation effect of the ligand from the metal complex, and depending on the spectroscopic properties of ligand and complex. The results regarding complex formation are compared to each other. Comparison of pairs by MALDI-TOF- and ESI-MS allows an estimation of the rank order of complexation tendency of one ligand with different metal ions and requires the least amount of substance. The other investigated methods provided additional information on structural properties of the formed complexes; however either they required too much pseudopeptide ligand or were not applicable for all transition metal ions used in this study.     

Contributions of five secondary metal uptake systems to metal homeostasis of Cupriavidus metallidurans CH34

Cupriavidus metallidurans is adapted to high concentrations of transition metal cations and is a model system for studying metal homeostasis in difficult environments. The elemental composition of C. metallidurans cells cultivated under various conditions was determined, revealing the ability of the bacterium to shield homeostasis of one essential metal from the toxic action of another. The contribution of metal uptake systems to this ability was studied. C. metallidurans contains three CorA members of the metal inorganic transport (MIT) protein family of putative magnesium uptake systems, ZupT of the ZRT/IRT protein, or ZIP, family, and PitA, which imports metal phosphate complexes. Expression of the genes for all these transporters was regulated by zinc availability, as shown by reporter gene fusions. While expression of zupT was upregulated under conditions of zinc starvation, expression of the other genes was downregulated at high zinc concentrations. Only corA(1) expression was influenced by magnesium starvation. Deletion mutants were constructed to characterize the contribution of each system to transition metal import. This identified ZupT as the main zinc uptake system under conditions of low zinc availability, CorA(1) as the main secondary magnesium uptake system, and CorA(2) and CorA(3) as backup systems for metal cation import. PitA may function as a cation-phosphate uptake system, the main supplier of divalent metal cations and phosphate in phosphate-rich environments. Thus, metal homeostasis in C. metallidurans is achieved by highly redundant metal uptake systems, which have only minimal cation selectivity and are in combination with efflux systems that "worry later" about surplus cations.     

Design of high affinity cyclic pentapeptide ligands for kappa-opioid receptors.

Using results from our previously reported cyclic opioid peptide series and reliable models for mu-, delta-, and kappa-opioid receptors (MOR, DOR, and KOR, respectively) and their complexes with peptide ligands, we have designed and synthesized a series of cyclic pentapeptides of structure Tyr-C[D-Cys-Phe-Phe-X]-NH2, cyclized via disulfide, methylene, or ethylene dithioethers, and where X = D- or L-Cys; or D- or L-penicillamine (Pen; beta,beta-dimethylcysteine). Determination of binding affinities to MOR, DOR, and KOR revealed that members of this series with X = D- or L-Cys display KOR affinities in the low nanomolar range, demonstrating that a 'DPDPE-like' tetrapeptide scaffold is suitable not only for DOR and MOR ligands, but also for KOR ligands. The cyclic pentapeptides reported here are not, however, selective for KOR, rather they display significant selectivity and high affinity for MOR. Indeed, peptide 8, Tyr-C[D-Cys-Phe-Phe-Cys]-NH2-cyclized via a methylene dithioether, shows picomolar binding affinity for MOR ( = 16 pm) with more than 100-fold selectivity for MOR vs. DOR or KOR, and may be of interest as a high affinity, high selectivity MOR ligand. Nonetheless, the high affinity KOR peptides in this series represent excellent leads for the development of structurally related, selective KOR ligands designed to exploit structurally specific features of KOR, MOR, and DOR.     

Differential effects of some transition metal cations on the binding of β-carboline-3-carboxylate and diazepam

The interaction of benzodiazepines and beta-carbolines with metal cations was investigated. Among numerous transition metal cations, only three, CO2+, Ni2+ and Zn2+, specifically inhibited the binding of [3H]beta-carboline-3-carboxylate ethyl ester (beta-CCE). The effects of these cations on [3H]beta-CCE binding were exactly opposite to those on [3H]diazepam binding. The effects of these cations was not dependent on lipid peroxidation. The differential effect of these cations may reflect a general difference in the way agonists and antagonists bind to the benzodiazepine receptor.     

Structures of the mono- and divalent metal nucleotide complexes in the myosin ATPase

The structure of both the mono- and the divalent metal nucleotide complexes active in the myosin subfragment 1 ATPase has been determined using the phosphorothioate analogs of ATP in the presence of various cations. Both the Sp and the Rp diastereomers of adenosine 5'-O-(1-thiotriphosphate) (ATP alpha S) were substrates in the presence of Mg2+, Ca2+, Mn2+, Co2+, Zn2+, and Cd2+ as well as with NH4+ and T1+. The Sp/Rp activity ratios obtained were largely independent of the cation. The simplest explanation of these results is that both mono- and divalent cations do not coordinate to the alpha-phosphate group. With adenosine 5'-O-(2-thiotriphosphate) (ATP beta S), essentially only the Sp diastereomer was active with Mg2+ with Sp/Rp ratio of greater 3000. As the divalent metal ion was varied in the series given above, this ratio was progressively lowered to the value of 0.2 found with Cd2+. Similar changes in stereoselectivity were seen with monovalent cations. Thus, with NH4+, an Sp/Rp ratio of 8 was observed, whereas with T1+, this figure was reduced to 0.04. These data indicate that both mono- and divalent cations coordinate to the beta-phosphate group of the nucleoside triphosphate substrate. These results obtained with ATP alpha S and ATP beta S suggest that myosin uses the mono- or divalent cation delta, beta, gamma-bidentate nucleotide chelate as substrate.