Interaction of mithramycin with chromatin

Mithramycin (MTR) is an anti-cancer antibiotic that blocks the macromolecular biosynthesis via reversible interaction with DNA template in the presence of bivalent metal ion such as Mg2+. In absence of DNA, mithramycin forms two types of complexes with Mg2+, complex I (with 1:1 stoichiometry in terms of MTR: Mg2+) and complex II (with 1:2 stoichiometry in terms of MTR: Mg2+). In an eukaryotic system, the drug would interact with chromatin, a protein-DNA complex. We have employed the spectroscopic techniques such as absorption and fluorescence to study the interaction of MTR: Mg2+ complexes with rat liver chromatin. In this report, we have shown that the two types of ligands have different binding potentials with the same chromatin. This supports our proposition that complexes I and II, are different molecular species. We have also shown that the histone protein(s) reduce the binding potential and the number of available sites for both ligands.     

Role of magnesium ion in the interaction between chromomycin A3 and DNA: binding of chromomycin A3-Mg2+ complexes with DNA.

Chromomycin A3 is an antitumor antibiotic which blocks macromolecular synthesis via reversible interaction with DNA template only in the presence of divalent metal ions such as Mg2+. The role of Mg2+ in this antibiotic-DNA interaction is not well understood. We approached the problem in two steps via studies on the interaction of (i) chromomycin A3 and Mg2+ and (ii) chromomycin A3-Mg2+ complex(es) and DNA. Spectroscopic techniques such as absorption, fluorescence, and CD were employed for this purpose. The results could be summed up in two parts. Absorption, fluorescence, and CD spectra of the antibiotic change upon addition of Mg2+ due to complex formation between them. Analysis of the quantitative dependence of change in absorbance of chromomycin A3 (at 440 nm) upon input concentration of Mg2+ indicates formation of two types of complexes with different stoichiometries and formation constants. Trends in change of fluorescence and CD spectroscopic features of the antibiotic in the presence of Mg2+ at different concentrations further corroborate this result. The two complexes are referred to as complex I (with 1:1 stoichiometry in terms of chromomycin A3:Mg2+) and complex II (with 2:1 stoichiometry in terms of chromomycin A3:Mg2+), respectively, in future discussions. The interactions of these complexes with calf thymus DNA were examined to check whether they bind differently to the same DNA. Evaluation of binding parameters, intrinsic binding constants, and binding stoichiometry, by means of spectrophotometric and fluorescence titrations, shows that they are different. Distinctive spectroscopic features of complexes I and II, when they are bound to DNA, also support that they bind differently to the above DNA. Measurement of thermodynamic parameters characterizing their interactions with calf thymus DNA shows that complex I-DNA interaction is exothermic, in contrast to complex II-DNA interaction, which is endothermic. This feature implies a difference in the molecular nature of the interactions between the complexes and calf thymus DNA. These observations are novel and significant to understand the antitumor property of the antibiotic. They are also discussed to provide explanations for the earlier reports that in some cases appeared to be contradictory.     

Interactions of chromomycin A3 and mithramycin with the sequence d(TAGCTAGCTA)2

Anti-cancer antibiotics, chromomycin A3 (CHR) and mithramycin (MTR) inhibit DNA directed RNA synthesis in vivo by binding reversibly to template DNA in the minor groove with GC base specificity, in the presence of divalent cations like Mg2+. Under physiological conditions, (drug)2Mg2+ complexes formed by the antibiotics are the potential DNA binding ligands. Structures of CHR and MTR differ in their saccharide residues. Scrutiny of the DNA binding properties reveal significant differences in their sequence selectivity, orientation and stoichiometry of binding. Here, we have analyzed binding and thermodynamic parameters for the interaction of the antibiotics with a model oligonucleotide sequence, d(TAGCTAGCTA)2 to understand the role of sugars. The oligomer contains two potential binding sites (GpC) for the ligands. The study illustrates that the drugs bind differently to the sequence. (MTR)2Mg2+ binds to both sites whereas (CHR)2Mg2+ binds to a single site. UV melting profiles for the decanucleotide saturated with the ligands show that MTR bound oligomer is highly stabilized and melts symmetrically. In contrast, with CHR, loss of symmetry in the oligomer following its association with a single (CHR)2Mg2+ complex molecule leads to a biphasic melting curve. Results have been interpreted in the light of saccharide dependent differences in ligand flexibility between the two antibiotics.     

Inhibition of a Zn(II)-containing enzyme, alcohol dehydrogenase, by anticancer antibiotics, mithramycin and chromomycin A3

One of the major attributes for the biological action of the aureolic acid anticancer antibiotics chromomycin A₃ (CHR) and mithramycin (MTR) is their ability to bind bivalent cations such as Mg(II) and Zn(II) ions and form high affinity 2:1 complexes in terms of the antibiotic and the metal ion, respectively. As most of the cellular Zn(II) ion is found to be associated with proteins, we have examined the effect of MTR/CHR on the structure and function of a representative structurally well characterized Zn(II) metalloenzyme, alcohol dehydrogenase (ADH) from yeast. MTR and CHR inhibit enzyme activity of ADH with inhibitory constants of micromolar order. Results from size-exclusion column chromatography, dynamic light scattering, and isothermal titration calorimetry have suggested that the mechanism of inhibition of the metalloenzyme by the antibiotics is due to the antibiotic-induced disruption of the enzyme quaternary structure. The nature of the enzyme inhibition, the binding stoichiometry of two antibiotics per monomer, and comparable dissociation constants for the antibiotic and free (or substrate-bound) ADH imply that the association occurs as a consequence of the binding of the antibiotics to Zn(II) ion present at the structural center. Confocal microscopy shows the colocalization of the antibiotic and the metalloenzyme in HepG2 cells, thereby supporting the proposition of physical association between the antibiotic(s) and the enzyme inside the cell.     

Structural and biochemical characterization of a new Mg(2+) binding site near Tyr94 in the restriction endonuclease PvuII

We have determined the crystal structure of the PvuII endonuclease in the presence of Mg(2+). According to the structural data, divalent metal ion binding in the PvuII subunits is highly asymmetric. The PvuII-Mg(2+) complex has two distinct metal ion binding sites, one in each monomer. One site is formed by the catalytic residues Asp58 and Glu68, and has extensive similarities to a catalytically important site found in all structurally examined restriction endonucleases. The other binding site is located in the other monomer, in the immediate vicinity of the hydroxyl group of Tyr94; it has no analogy to metal ion binding sites found so far in restriction endonucleases. To assign the number of metal ions involved and to better understand the role of Mg(2+) binding to Tyr94 for the function of PvuII, we have exchanged Tyr94 by Phe and characterized the metal ion dependence of DNA cleavage of wild-type PvuII and the Y94F variant. Wild-type PvuII cleaves both strands of the DNA in a concerted reaction. Mg(2+) binding, as measured by the Mg(2+) dependence of DNA cleavage, occurs with a Hill coefficient of 4, meaning that at least two metal ions are bound to each subunit in a cooperative fashion upon formation of the active complex. Quenched-flow experiments show that DNA cleavage occurs about tenfold faster if Mg(2+) is pre-incubated with enzyme or DNA than if preformed enzyme-DNA complexes are mixed with Mg(2+). These results show that Mg(2+) cannot easily enter the active center of the preformed enzyme-DNA complex, but that for fast cleavage the metal ions must already be bound to the apoenzyme and carried with the enzyme into the enzyme-DNA complex. The Y94F variant, in contrast to wild-type PvuII, does not cleave DNA in a concerted manner and metal ion binding occurs with a Hill coefficient of 1. These results indicate that removal of the Mg(2+) binding site at Tyr94 completely disrupts the cooperativity in DNA cleavage. Moreover, in quenched-flow experiments Y94F cleaves DNA about ten times more slowly than wild-type PvuII, regardless of the order of mixing. From these results we conclude that wild-type PvuII cleaves DNA in a fast and concerted reaction, because the Mg(2+) required for catalysis are already bound at the enzyme, one of them at Tyr94. We suggest that this Mg(2+) is shifted to the active center during binding of a specific DNA substrate. These results, for the first time, shed light on the pathway by which metal ions as essential cofactors enter the catalytic center of restriction endonucleases.     

Thermodynamic linked-function analysis of Mg(2+)-activated yeast pyruvate kinase.

Yeast pyruvate kinase (YPK) is regulated by intermediates of the glycolytic pathway [e.g., phosphoenolpyruvate (PEP), fructose 1,6-bisphosphate (FBP), and citrate] and by the ATP charge of the cell. Recent kinetic and thermodynamic data with Mn(2+)-activated YPK show that Mn(2+) mediates the allosteric communication between the substrate, PEP, and the allosteric effector, FBP [Mesecar, A., and Nowak, T. (1997) Biochemistry 36, 6792, 6803]. These results indicate that divalent cations modulate multiligand interactions, and hence cooperativity with YPK. The nature of multiligand interactions on YPK was investigated in the presence of the physiological divalent activator Mg(2+). The binding interactions of PEP, Mg(2+), and FBP were monitored by fluorescence spectroscopy. The binding data were subject to thermodynamic linked-function analysis to determine the magnitudes of the multiligand interactions governing the allosteric activation of YPK. The two ligand coupling free energies between PEP and Mg(2+), PEP and FBP, and FBP and Mg(2+) are 0.88, -0.38, and -0.75 kcal/mol, respectively. The two-ligand coupling free energies between PEP and Mn(2+) and FBP and Mn(2+) are more negative than those with Mg(2+) as the cation. This indicates that the interactions between the divalent cation and PEP with YPK are different for Mg(2+) and Mn(2+) and that the interaction is not simply electrostatic in nature, as originally hypothesized. The magnitude of the heterotropic interaction between the metal and FBP is similar with Mg(2+) and Mn(2+). The simultaneous binding of Mg(2+), PEP, and FBP to YPK is favored by 3.21 kcal/mol compared to independent binding. This complex is destabilized by 3.30 kcal/mol relative to the analogous YPK-Mn(2+)-PEP-FDP complex. Interpretation of K(d) values when cooperative binding occurs must be done with care as these are not simple thermodynamic constants. These data demonstrate that the divalent metal, which activates phosphoryl transfer in YPK, plays a key role in modulating the various multiligand interactions that define the overall allosteric properties of the enzyme.     

The second Ca(2+)-binding domain of NCX1 binds Mg2+ with high affinity

We report the effects of binding of Mg(2+) to the second Ca(2+)-binding domain (CBD2) of the sodium-calcium exchanger. CBD2 is known to bind two Ca(2+) ions using its Ca(2+)-binding sites I and II. Here, we show by nuclear magnetic resonance (NMR), circular dichroism, isothermal titration calorimetry, and mutagenesis that CBD2 also binds Mg(2+) at both sites, but with significantly different affinities. The results from Mg(2+)-Ca(2+) competition experiments show that Ca(2+) can replace Mg(2+) from site I, but not site II, and that Mg(2+) binding affects the affinity for Ca(2+). Furthermore, thermal unfolding circular dichroism data demonstrate that Mg(2+) binding stabilizes the domain. NMR chemical shift perturbations and (15)N relaxation data reveal that Mg(2+)-bound CBD2 adopts a state intermediate between the apo and fully Ca(2+)-loaded forms. Together, the data show that at physiological Mg(2+) concentrations CBD2 is loaded with Mg(2+) preferentially at site II, thereby stabilizing and structuring the domain and altering its affinity for Ca(2+).     

Mg(2+) in the Major Groove Modulates B-DNA Structure and Dynamics

This study investigates the effect of Mg(2+) bound to the DNA major groove on DNA structure and dynamics. The analysis of a comprehensive dataset of B-DNA crystallographic structures shows that divalent cations are preferentially located in the DNA major groove where they interact with successive bases of (A/G)pG and the phosphate group of 5'-CpA or TpG. Based on this knowledge, molecular dynamics simulations were carried out on a DNA oligomer without or with Mg(2+) close to an ApG step. These simulations showed that the hydrated Mg(2+) forms a stable intra-strand cross-link between the two purines in solution. ApG generates an electrostatic potential in the major groove that is particularly attractive for cations; its intrinsic conformation is well-adapted to the formation of water-mediated hydrogen bonds with Mg(2+). The binding of Mg(2+) modulates the behavior of the 5'-neighboring step by increasing the BII (ε-ζ>0°) population of its phosphate group. Additional electrostatic interactions between the 5'-phosphate group and Mg(2+) strengthen both the DNA-cation binding and the BII character of the 5'-step. Cation binding in the major groove may therefore locally influence the DNA conformational landscape, suggesting a possible avenue for better understanding how strong DNA distortions can be stabilized in protein-DNA complexes.     

Thermodynamics of cation binding to rabbit skeletal muscle calsequestrin. Evidence for distinct Ca(2+)- and Mg(2+)-binding sites

Ca2+ binding to rabbit skeletal calsequestrin was studied at physiological ionic strength by equilibrium flow dialysis, Hummel-Dryer gel filtration and microcalorimetry. 31 Ca(2+)-binding sites with a mean dissociation constant (KD) of 0.79 mM were titrated in the absence, and 23 sites with a KD of 0.88 mM in the presence of 3 mM Mg2+. No cooperativity was observed. For Mg2+ binding, the combination of gel filtration and microcalorimetry yielded a stoichiometry of 26 Mg2+/protein with a KD of 2mM. 1 mM Ca2+ decreased the stoichiometry to 20 Mg2+/protein. Binding of Ca2+ in the absence and presence of 3 mM Mg2+ was accompanied by a release of 2.0 and 2.7 H+/protein, respectively. Mg2+ binding did not lead to a significant proton release suggesting a qualitative difference in the Ca(2+)- and Mg(2+)-binding sites. After correction for proton release, the enthalpy change for Ca2+ binding was very low (-1.5 kJ/protein in the absence, and -15 kJ/protein in the presence of 3 mM Mg2+). The entropy change (+59 J/K.site in the absence and +56 J/K.site in the presence of Mg2+) was therefore virtually the sole driving force for Ca2+ binding. Mg2+ binding is slightly more exothermic (-12.6 kJ/protein), but as for Ca2+, the entropy change (+50 J/K.site) constituted the major driving force of the reaction. A fluorimetric study indicates that the conformation of tryptophan in Mg(2+)-saturated calsequestrin was clearly different from that in the Ca(2+)-saturated protein, but that the (Ca2+ + Mg2+)-saturated protein was not distinct from the Ca(2+)-saturated protein. Thus, in addition to the thermodynamic characterization of the Ca2+/calsequestrin interaction, our data indicate that Ca2+ and Mg2+ do not bind to the same sites on calsequestrin. The data also predict considerable proton fluxes upon Ca(2+)-Mg2+ exchange in vivo.     

RC3/Neurogranin and Ca2+/calmodulin-dependent protein kinase II produce opposing effects on the affinity of calmodulin for calcium

The interaction of calmodulin with its target proteins is known to affect the kinetics and affinity of Ca(2+) binding to calmodulin. Based on thermodynamic principles, proteins that bind to Ca(2+)-calmodulin should increase the affinity of calmodulin for Ca(2+), while proteins that bind to apo-calmodulin should decrease its affinity for Ca(2+). We quantified the effects on Ca(2+)-calmodulin interaction of two neuronal calmodulin targets: RC3, which binds both Ca(2+)- and apo-calmodulin, and alphaCaM kinase II, which binds selectively to Ca(2+)-calmodulin. RC3 was found to decrease the affinity of calmodulin for Ca(2+), whereas CaM kinase II increases the calmodulin affinity for Ca(2+). Specifically, RC3 increases the rate of Ca(2+) dissociation from the C-terminal sites of calmodulin up to 60-fold while having little effect on the rate of Ca(2+) association. Conversely, CaM kinase II decreases the rates of dissociation of Ca(2+) from both lobes of calmodulin and autophosphorylation of CaM kinase II at Thr(286) induces a further decrease in the rates of Ca(2+) dissociation. RC3 dampens the effects of CaM kinase II on Ca(2+) dissociation by increasing the rate of dissociation from the C-terminal lobe of calmodulin when in the presence of CaM kinase II. This effect is not seen with phosphorylated CaM kinase II. The results are interpreted according to a kinetic scheme in which there are competing pathways for dissociation of the Ca(2+)-calmodulin target complex. This work indicates that the Ca(2+) binding properties of calmodulin are highly regulated and reveals a role for RC3 in accelerating the dissociation of Ca(2+)-calmodulin target complexes at the end of a Ca(2+) signal.     

Chelation of divalent cations by ATP, studied by titration calorimetry.

Thermodynamic parameters and stoichiometry for the formation of complexes of ATP with Mg2+, Ca2+, and Sr2+ were determined by titration calorimetry. In each case, 1:1 stoichiometry was observed and complex formation was entropy driven. Binding constants for formation of complexes decreased in the order of Mg2+ greater than Ca2+ greater than Sr2+, as expected from charge density considerations. Monovalent cations hindered complex formation with Mg2+, apparently by competing with the divalent cation for complexation with ATP. Analysis of this competitive effect provided estimates of the binding constants for complexes of ATP with monovalent cations, which decreased in the order expected from charge density considerations (Li+ greater than Na+ greater than K+).     

Determinants of Mg2+-dependent activities of recombinant human immunodeficiency virus type 1 integrase

The relationship between Mg(2+)-dependent activity and the self-assembly state of HIV-1 integrase was investigated using different protein preparations. The first preparations, IN(CHAPS) and IN(dial), were purified in the presence of detergent, but in the case of IN(dial), the detergent was removed during a final dialysis. The third preparation, IN(zn), was purified without any detergent. The three preparations displayed comparable Mn(2+)-dependent activities. In contrast, the Mg(2+)-dependent activity that reflects a more realistic view of the physiological activity strongly depended on the preparation. IN(CHAPS) was not capable of using Mg(2+) as a cofactor, whereas IN(zn) was highly active under the same conditions. In the accompanying paper [Deprez, E., et al. (2000) Biochemistry 39, 9275-9284], we used time-resolved fluorescence anisotropy to demonstrate that IN(CHAPS) was monomeric at the concentration of enzymatic assays. Here, we show that IN(zn) was homogeneously tetrameric under similar conditions. Moreover, IN(dial) that exhibited an intermediary Mg(2+)-dependent activity existed in a monomer-multimer equilibrium. The level of Mg(2+)- but not Mn(2+)-dependent activity of IN(dial) was altered by addition of detergent which plays a detrimental role in the maintenance of the oligomeric organization. Our results indicate that the ability of integrase to use Mg(2+) as a cofactor is related to its self-assembly state in solution, whereas Mn(2+)-dependent activity is not. Finally, the oligomeric IN(zn) was capable of binding efficiently to DNA regardless of the cationic cofactor, whereas the monomeric IN(CHAPS) strictly required Mn(2+). Thus, we propose that a specific conformation of integrase is a prerequisite for its binding to DNA in the presence of Mg(2+).