Electrostatic potential of aminoacyl-tRNA synthetase navigates tRNA on its pathway to the binding site

In the first stage of a diffusion-controlled enzymatic reaction, aminoacyl-tRNA synthetases (aaRSs) interact with cognate tRNAs forming non-specific encounters. The aaRSs catalyzing the same overall aminoacylation reaction vary greatly in subunit organization, structural domain composition and amino acid sequence. The diffusional association of aaRS and tRNA was found to be governed by long-range electrostatic interactions when the homogeneous negative potential of tRNA fits to the patches of positive potential produced by aaRS; one patch for each tRNA substrate molecule. Considering aaRS as a molecule with anisotropic reactivity and on the basis of continuum electrostatics and Smoluchowski's theory, the reaction conditions for tRNA-aaRS diffusional encounters were formulated. The domains, categorized as enzymatically relevant, appeared to be non-essential for field sculpturing at long distances. On the other hand, a set of complementary domains exerts primary control on the aaRS isopotential surface formation. Subdividing the aaRS charged residues into native, conservative and non-conservative subsets, we evaluated the contribution of each group to long-range electrostatic potential. Surprisingly, the electrostatic potential landscapes generated by native and non-conservative subsets are fairly similar, thus suggesting the non-conservative subset is developed specifically for efficient tRNA attraction.     

Role of divalent ions in folding of tRNA.

The native structure of tRNA is not achieved in low salt (4.5 mM Na+, 25 degrees C), but can be restored by addition of divalent ions. We have explored the structure of the central region in Escherichia coli tRNAfMet by absorption and emission spectroscopy of 4-thiouracil, and the structure of the anticodon loop in yeast tRNAPhe by fluorescence of the 'Y' base, versus the number of manganese ions bound to tRNA, which was derived from electron spin resonance. The fluorescence of the reduced 8-13 photoproduct (in which 4-thiouracil at position 8 is crosslinked to cytosine at position 13) was also analysed. In low salt (e.g. 4.5 mM Na+), the region of 4-thiouracil is affected strongly as the first eight Mn2+ bind to tRNA, whereas the fluorescence of the 'Y' base is affected only after four Mn2+ are bound. Considering the structural similarities of the two tRNAs, this suggests that the reorganisation brought about by divalent ions starts in the central region, the anticodon loop being affected later. The binding of divalent ions to each region starts together with its restructuration. Monovalent ions can substitute for divalent ions in this process, a 15 mM sodium concentration being equivalent to the binding of the first five Mn2+. If divalent ions are then added, even the first ones distribute themselves between both the central and the anticodon region. Alternatively, the renaturation may be achieved by monovalent ions only, implying that no sites exist whose occupancy by divalent ions is crucial for the native structure. These observations suggest that the role and means of divalent ion binding to tRNA are largely explainable in terms of a simple maganese-phosphate binding supplemented by electrostatic interaction with distant phosphates.     

Lead ion binding and RNA chain hydrolysis in phenylalanine tRNA

Crystalline complexes of yeast phenylalanine tRNA and Lead (II) ion were prepared by soaking pregrown orthorhombic crystals of tRNA in saturated lead chloride solutions. The locations of tightly bound lead ions on the tRNA were determined by difference Fourier methods. There are three major lead binding sites; two of these replace tightly bound magnesium ions in the native tRNA structure. Site I is located in the dihydrouridine loop of the molecule adjacent to phosphate P18 which is specifically cleaved by lead. This is evident from changes observed in the Pb-native difference electron density maps. A possible mechanism for lead ion hydrolysis of the polynucleotide chain is proposed.     

Electrostatic effects on the stability of condensed DNA in the presence of divalent cations.

Cylindrical cell model Poisson-Boltzmann (P-B) calculations are used to evaluate the electrostatic contributions to the relative stability of various DNA conformations (A, B, C, Z, and single-stranded (ss) with charge spacings of 3.38 and 4.2 A) as a function of interhelix distance in a concentrated solution of divalent cations. The divalent ion concentration was set at 100 mM, to compare with our earlier reports of spectroscopic and calorimetric experiments, which demonstrate substantial disruption of B-DNA geometry. Monovalent cations neutralize the DNA phosphates in two ways, corresponding to different experimental situations: 1) There is no significant contribution to the ionic strength from the neutralizing cations, corresponding to DNA condensation from dilute solution and to osmotic stress experiments in which DNA segments are brought into close proximity to each other in the presence of a large excess of buffer. 2) The solution is uniformly concentrated in DNA, so that the neutralizing cations add significantly to those in the buffer at close DNA packing. In case 1), conformations with lower charge density (Z and ssDNA) have markedly lower electrostatic free energies than B-DNA as the DNA molecules approach closely, due largely to ionic entropy. If the divalent cations bind preferentially to single-stranded DNA or a distorted form of B-DNA, as is the case with transition metals, the base pairing and stacking free energies that stabilize the double helix against electrostatic denaturation may be overcome. Strong binding to the bases is favored by the high concentration of divalent cations at the DNA surface arising from the large negative surface potential; the surface concentration increases sharply as the interhelical distance decreases. In case 2), the concentration of neutralizing monovalent cations becomes very large and the electrostatic free energy difference between secondary structures becomes small as the interhelical spacing decreases. Such high ionic concentrations will be expected to modify the stability of DNA by changing water activity as well as by screening electrostatic interactions. This may be the root of the decreased thermal stability of DNA in the presence of high concentrations of magnesium ions.     

The binding of divalent cations to myosin.

Centrifuge transport, equilibrium dialysis, and electron paramagnetic resonance studies on the binding of Mn2+ to myosin revealed two sets of noninteracting binding sites which are characterized at low ionic strength (0.016 M KCl) by affinity constants of 10(6) M-1 (Class I) and 10(3) M-1 (Class II), respectively. At 0.6 M KCl concentration, the affinity of Mn2+ for both sets of sites is reduced. The maximum number of binding sites is 2 for the high affinity and 20 to 25 for the low affinity set. Other divalent metal ions displace Mn2+ from the high affinity sites in the following order of effectiveness: Ca greater than Mg = Zn = Co greater than Sr greater than Ni. The inhibitory effects of Mg2+ and Ca2+ upon the Mn2+ binding are competitive with inhibitor constants of 0.75 to 1 mM which is similar to that of the low affinity divalent metal ion binding sites. Exposure of myosin to 37 degrees partially inhibits Mn2+ binding to Class I parallel with inhibition of ATPase activity. The binding of Mn2+ to the high affinity binding sites is not significantly influenced by ADP or PPi, although Mn2+ increases the affinity of ADP binding to myosin at high ionic strength.     

Aminoglycoside binding displaces a divalent metal ion in a tRNA-neomycin B complex

Aminoglycosides bind to RNA and interfere with its function, and it has been suggested that aminoglycoside binding to RNA displaces essential divalent metal ions. Here we demonstrate that addition of various aminoglycosides inhibited Pb2+-induced cleavage of yeast tRNA(Phe). Cocrystallization of yeast tRNA(Phe) and an aminoglycoside, neomycin B, resulted in crystals that diffracted to 2.6 A and the structure of the complex was solved by molecular replacement. The structure shows that the neomycin B binding site overlaps with known divalent metal ion binding sites in yeast tRNA(Phe), providing direct evidence for the hypothesis that aminoglycosides displace metal ions. Additionally, the neomycin B binding site overlaps with major determinants for Escherichia coli phenylalanyl-tRNA-synthetase. Here we present data demonstrating that addition of neomycin B inhibited aminoacylation of E. coli tRNA(Phe) in the mid microM range. Given that aminoglycoside and metal ion binding sites overlap, we discuss that aminoglycosides can be considered as 'metal mimics'.     

Mechanistic studies on E. coli DNA topoisomerase I: divalent ion effects.

E. coli DNA topoisomerase I catalyzes the hydrolysis of short, single stranded oligodeoxynucleotides. It also forms a covalent protein-DNA complex with negatively supercoiled DNA in the absence of Mg2+ but requires Mg2+ for the relaxation of negatively supercoiled DNA. In this paper we investigate the effects of various divalent metals on catalysis. For the relaxation reaction, maximum enzyme activity plateaus after 2.5 mM Mg2+. However, the rate of cleavage of short oligodeoxynucleotide increased linearly between 0 and 15 mM Mg2+. In the oligodeoxynucleotide cleavage reaction, Ca2+, Mn2+, Co2+, and Zn2+ inhibit enzymatic activity. When these metals are coincubated with Mg2+ at equimolar concentrations, the normal effect of Mg2+ is not detectable. Of these metals, only Ca2+ can be substituted for Mg2+ as a metal cofactor in the relaxation reaction. And when Mg2+ is coincubated with Mn2+, Co2+, or Zn2+ at equimolar concentrations, the normal effect of Mg2+ on relaxation is not detectable. We propose that Mg2+ allows the protein-DNA complex to assume a conformation necessary for strand passage and enhance the rate of enzyme turnover.     

Solution structure and thermodynamics of a divalent metal ion binding site in an RNA pseudoknot.

Identification and characterization of a metal ion binding site in an RNA pseudoknot was accomplished using cobalt (III) hexammine, Co(NH3)63+, as a probe for magnesium (II) hexahydrate, Mg(H2O)62+, in nuclear magnetic resonance (NMR) structural studies. The pseudoknot causes efficient -1 ribosomal frameshifting in mouse mammary tumor virus. Divalent metal ions, such as Mg2+, are critical for RNA structure and function; Mg2+preferentially stabilizes the pseudoknot relative to its constituent hairpins. The use of Co(NH3)63+as a substitute for Mg2+was investigated by ultraviolet absorbance melting curves, NMR titrations of the imino protons, and analysis of NMR spectra in the presence of Mg2+or Co (NH3)63+. The structure of the pseudoknot-Co(NH3)63+complex reveals an ion-binding pocket formed by a short, two-nucleotide loop and the major groove of a stem. Co(NH3)63+stabilizes the sharp loop-to-stem turn and reduces the electrostatic repulsion of the phosphates in three proximal strands. Hydrogen bonds are identified between the Co(NH3)63+protons and non-bridging phosphate oxygen atoms, 2' hydroxyl groups, and nitrogen and oxygen acceptors on the bases. The binding site is significantly different from that previously characterized in the major groove surface of tandem G.U base-pairs, but is similar to those observed in crystal structures of a fragment of the 5 S rRNA and the P5c helix of the Tetrahymena thermophila group I intron. Changes in chemical shifts occurred at the same pseudoknot protons on addition of Mg2+as on addition of Co(NH3)63+, indicating that both ions bind at the same site. Ion binding dissociation constants of approximately 0.6 mM and 5 mM (in 200 mM Na+and a temperature of 15 degrees C) were obtained for Co(NH3)63+and Mg2+, respectively, from the change in chemical shift as a function of metal ion concentration. An extensive array of non-sequence-specific hydrogen bond acceptors coupled with conserved structural elements within the binding pocket suggest a general mode of divalent metal ion stabilization of this type of frameshifter pseudoknot. These results provide new thermodynamic and structural insights into the role divalent metal ions play in stabilizing RNA tertiary structural motifs such as pseudoknots.     

Electrostatic coupling of ion pumps.

In this paper the electrostatic interactions between membrane-embedded ion-pumps and their consequences for the kinetics of pump-mediated transport processes have been examined. We show that the time course of an intrinsically monomolecular transport reaction can become distinctly nonexponential, if the reaction is associated with charge translocation and takes place in an aggregate of pump molecules. First we consider the electrostatic coupling of a single dimer of ion-pumps embedded in the membrane. Then we apply the treatment to the kinetic analysis of light-driven proton transport by bacteriorhodopsin which forms two-dimensional hexagonal lattices. Finally, for the case of nonordered molecules, we also consider a model in which the pumps are randomly distributed over the nodes of a lattice. Here the average distance is equal to that deduced experimentally and the elemental size of the lattice is the effective diameter of one single pump. This latter model is applied to an aggregate of membrane-embedded Na, K- and Ca-pumps. In all these cases the electrostatic potential considered is the exact solution calculated from the method of electrical images for a plane membrane of finite thickness immersed in an infinite aqueous solution environment. The distributions of charges (ions or charged binding sites) are considered homogeneous or discrete in the membrane and/or in the external solution. In the case of discrete distributions we compare the results from a mean field approximation and a stochastic simulation.     

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.     

Electrostatic effects in hemoglobin: hydrogen ion equilibria in human deoxy- and oxyhemoglobin A.

The modified Tanford-Kirkwood theory of Shire et al. [Shire, S. J., Hanania, G.I.H., & Gurd, F.R.N. (1974) Biochemistry 13, 2967] for electrostatic interactions was applied to the hydrogen ion equilibria of human deoxyhemoglobin and oxyhemoglobin. Atomic coordinates for oxyhemoglobin were generated by the application of the appropriate rigid rotation function to alpha and beta chains of the deoxyhemoglobin structure [Fermi, G. (1975) J. Mol. Biol. 97, 237]. The model employs two sets of parameters derived from the crystalline protein structures, the atomic coordinates of charged amino acid residues and static solvent accessibility factors to reflect their individual degrees of exposure to solvent. Theoretical titration curves based on a consistent set of pKint values compared closely with experimental potentiometric curves. Theoretical pK values at half-titration for individual protein sites corresponded to available observed values for both quaternary states. The results bring out the cumulative effects of numerous electrostatic interactions in the tetrameric structures and the major effects of the quaternary transition that result from changes in static solvent accessibility of certain ionizable groups.     

Multibody effects in ion binding and selectivity

Selective binding of ions to biomolecules plays a vital role in numerous biological processes. To understand the specific role of induced effects in selective ion binding, we use quantum chemical and pairwise-additive force-field simulations to study Na⁺ and K⁺ binding to various small molecules representative of ion binding functional groups in biomolecules. These studies indicate that electronic polarization significantly contributes to both absolute and relative ion-binding affinities. Furthermore, this contribution depends on both the number and the specific chemistries of the coordinating molecules, thus highlighting the complexity of ion-ligand interactions. Specifically, multibody interactions reduce as well as enhance the dipole moments of the ion-coordinating molecules, thereby affecting observables like coordination number distributions of ions. The differential polarization induced in molecules coordinating these two equivalently charged, but different-sized, ions also depends upon the number of coordinating molecules, showing the importance of multibody effects in distinguishing these ions thermodynamically. Because even small differences in ionic radii (0.4 Å for Na⁺ and K⁺) produce differential polarization trends critical to distinguishing ions thermodynamically, it is likely that polarization plays an important role in thermodynamically distinguishing other ions and charged chemical and biological functional groups.     

Preferential binding of yeast tRNA ligase to pre-tRNA substrates.

Joining of tRNA halves during splicing in extracts of Saccharomyces cerevisiae requires each of the three enzymatic activities associated with the tRNA ligase polypeptide. Joining is most efficient for tRNA as opposed to oligonucleotide substrates and is sensitive to single base changes at a distance from splice sites suggesting considerable specificity. To examine the basis for this specificity, binding of ligase to labeled RNA substrates was measured by native gel electrophoresis. Ligase bound tRNA halves with an association constant 1600-fold greater than that for a nonspecific RNA. Comparison of binding of a series of tRNA processing intermediates revealed that tRNA-structure, particularly in the region around the splice sites, contributes to specific binding. Finally, the ligase was shown to form multiple, discrete complexes with tRNA substrates. The basis for recognition by ligase and its role in a tRNA processing pathway are discussed.     

High affinity divalent cation binding to actin. Effect of low affinity salt binding

Monomeric actin labeled with the fluorescent probe N-iodoacetyl-N'-(5-sulfo-1-naphthyl)ethylenediamine (1,5-I-AEDANS-actin) displays a fast fluorescence intensity increase immediately upon addition of salt and then a slow fluorescence intensity change concurrent with Ca2+/Mg2+ exchange at the high affinity divalent cation binding site on actin. The fast change appears to reflect competitive binding of K+ at low affinity (nonspecific) sites and of Mg2+ or Ca2+ at low and intermediate affinity sites. Binding of cation at the low affinity sites (but apparently not at the intermediate affinity sites) results in an increase in k-Ca and k-Mg and thus a decrease in affinity for divalent cations at the high affinity site. The effect of Mg2+ on k-Ca is twice that of K+ for equal fractional saturations of the low affinity binding, and the effect of K+ and Mg2+ together on k-Ca reflects competitive binding at the low affinity sites. Thus the affinity and kinetics of divalent cation binding at the high affinity site of actin are significantly affected by concurrent cation binding at low affinity sites.     

An energy dependent divalent cation-hydrogen ion binding in the outer membranes of rat liver mitochondria and its dependence on monovalent cation-hydrogen ion binding

Intact mitochondria are able to bind monovalent and divalent metal cations and to release protons in an energy-independent exchange process. Directly accessible binding sites exist in the outer membrane. They seem to be identical for monovalent and divalent metal ions. The inner membrane-matrix-fraction possesses exchange sites after ultrasonic disruption only for monovalent cations, but not for divalent cations.     

Electrostatic contributions to heat capacity changes of DNA-ligand binding.

Significant heat capacity changes (DeltaCp) often accompany protein unfolding, protein binding, and specific DNA-ligand binding reactions. Such changes are widely used to analyze contributions arising from hydrophobic and polar hydration. Current models relate the magnitude of DeltaCp to the solvent accessible surface area (ASA) of the molecule. However, for many binding systems-particularly those involving non-peptide ligands-these models predict a DeltaCp that is significantly different from the experimentally measured value. Electrostatic interactions provide a potential source of heat capacity changes and do not scale with ASA. Using finite-difference Poisson-Boltzmann methods (FDPB), we have determined the contribution of electrostatics to the DeltaCp associated with binding for DNA binding reactions involving the ligands DAPI, netropsin, lexitropsin, and the lambda repressor binding domain.