Binding of transition metal ions to albumin: Sites,affinities and rates

Background

Serum albumin is the most abundant protein in the blood and cerebrospinal fluid and plays a fundamental role in the distribution of essential transition metal ions in the human body. Human serum albumin (HSA) is an important physiological transporter of the essential metal ions Cu^2 +, and Zn^2 + in the bloodstream. Its binding of metals like Ni^2 +, Co^2 +, or Cd^2 + can occur in vivo, but is only of toxicological relevance. Moreover, HSA is one of the main targets and hence most studied binding protein for metallodrugs based on complexes with Au, Pt and V.

Scope of Review

We discuss i) the four metal-binding sites so far described on HSA, their localization and metal preference, ii) the binding of the metal ions mentioned above, i.e. their stability constants and association/dissociation rates, their coordination chemistry and their selectivity versus the four binding sites iii) the methodology applied to study issues of items i and ii and iv) oligopeptide models of the N-terminal binding site.

Major Conclusions

Albumin has four partially selective metal binding sites with well-defined metal preferences. It is an important regulator of the blood transport of physiological Cu(II) and Zn(II) and toxic Ni(II) and Cd(II). It is also an important target for metal-based drugs containing Pt(II), V(IV)O, and Au(I).

General Significance

The thorough understanding of metal binding properties of serum albumin, including the competition of various metal ions for specific binding sites is important for biomedical issues, such as new disease markers and design of metal-based drugs. This article is part of a Special Issue entitled Serum Albumin.     

The interaction of manganese ions with DNA

We present the structure of the duplex formed by a fragment of auto-complementary DNA with the sequence d(CGTTAATTAACG). The structure was determined by X-ray crystallography. Up to date it is the first structure presenting the interaction between a DNA oligonucleotide and manganese ions. The presence of Mn²⁺ creates bonds among the N7 atom of guanines and phosphates. These bonds stabilize and determine the crystallographic network in a P3₂ space group, unusual in oligonucleotide crystals. The crystal structure observed is compared with those found in the presence of Mg²⁺, Ca²⁺ and Ni²⁺, which show different kinds of interactions. The double helices show end-to-end interactions, in a manner that the terminal guanines interact with the minor groove of the neighboring duplex, while the terminal cytosines are disordered. We have chosen this sequence since it contains a TTAA repeat. Such repeats are very rare in all genomes. We suggest that this sequence may be very susceptible to the formation of closely spaced thymine dimers.     

Metal ions and phosphate binding in the H-N-H motif: crystal structures of the nuclease domain of ColE7/Im7 in complex with a phosphate ion and different divalent metal ions

H-N-H is a motif found in the nuclease domain of a subfamily of bacteria toxins, including colicin E7, that are capable of cleaving DNA nonspecifically. This H-N-H motif has also been identified in a subfamily of homing endonucleases, which cleave DNA site specifically. To better understand the role of metal ions in the H-N-H motif during DNA hydrolysis, we crystallized the nuclease domain of colicin E7 (nuclease-ColE7) in complex with its inhibitor Im7 in two different crystal forms, and we resolved the structures of EDTA-treated, Zn(2+)-bound and Mn(2+)-bound complexes in the presence of phosphate ions at resolutions of 2.6 A to 2.0 A. This study offers the first determination of the structure of a metal-free and substrate-free enzyme in the H-N-H family. The H-N-H motif contains two antiparallel beta-strands linked to a C-terminal alpha-helix, with a divalent metal ion located in the center. Here we show that the metal-binding sites in the center of the H-N-H motif, for the EDTA-treated and Mg(2+)-soaked complex crystals, were occupied by water molecules, indicating that an alkaline earth metal ion does not reside in the same position as a transition metal ion in the H-N-H motif. However, a Zn(2+) or Mn(2+) ions were observed in the center of the H-N-H motif in cases of Zn(2+) or Mn(2+)-soaked crystals, as confirmed in anomalous difference maps. A phosphate ion was found to bridge between the divalent transition metal ion and His545. Based on these structures and structural comparisons with other nucleases, we suggest a functional role for the divalent transition metal ion in the H-N-H motif in stabilizing the phosphoanion in the transition state during hydrolysis.     

Some effects of metal ions on DNA structure and genetic information transfer

The reaction of metal ions with nucleic acids can lead to a variety of dramatic effects on nucleic acid structure, e.g., crosslinking of the polymer strands, degradation to oligomers and monomers, stabilization or destabilization, and the mispairing of bases. These effects have important implications for genetic information transfer. Metal ions are involved in many aspects of this transfer; we are presently concerned with the effect of metal ions on the orientation of the active site of RNA polymerase. Many of the effects of metal ions on nucleic acid structure involve changes in the conformation of the macromolecules. We have found that conditions that have been used to convert B DNA to Z DNA lead to at least two other conformational changes, and phase diagrams delineate the realms of stability of each of the forms. We have carried out a number of studies that demonstrate that the conversion of B to Z DNA is very closely correlated with a substantial decrease in the ability of the DNA to act as a template for RNA synthesis. A portion of this paper has been taken from another paper on “Changes of Biological Significance Induced by Metal Ions in the Structure of Nucleic Acids,” published in Annali dell' lstituto Superiore di Sanita.     

Uncoupling metallonuclease metal ion binding sites via nudge mutagenesis

The hydrolysis of phosphodiester bonds by nucleases is critical to nucleic acid processing. Many nucleases utilize metal ion cofactors, and for a number of these enzymes two active-site metal ions have been detected. Testing proposed mechanistic roles for individual bound metal ions has been hampered by the similarity between the sites and cooperative behavior. In the homodimeric PvuII restriction endonuclease, the metal ion dependence of DNA binding is sigmoidal and consistent with two classes of coupled metal ion binding sites. We reasoned that a conservative active-site mutation would perturb the ligand field sufficiently to observe the titration of individual metal ion binding sites without significantly disturbing enzyme function. Indeed, mutation of a Tyr residue 5.5 A from both metal ions in the enzyme-substrate crystal structure (Y94F) renders the metal ion dependence of DNA binding biphasic: two classes of metal ion binding sites become distinct in the presence of DNA. The perturbation in metal ion coordination is supported by 1H-15N heteronuclear single quantum coherence spectra of enzyme-Ca(II) and enzyme-Ca(II)-DNA complexes. Metal ion binding by free Y94F is basically unperturbed: through multiple experiments with different metal ions, the data are consistent with two alkaline earth metal ion binding sites per subunit of low millimolar affinity, behavior which is very similar to that of the wild type. The results presented here indicate a role for the hydroxyl group of Tyr94 in the coupling of metal ion binding sites in the presence of DNA. Its removal causes the affinities for the two metal ion binding sites to be resolved in the presence of substrate. Such tuning of metal ion affinities will be invaluable to efforts to ascertain the contributions of individual bound metal ions to metallonuclease function.     

Isothermal titration calorimetry of metal ions binding to proteins: An overview of recent studies

Isothermal titration calorimetry (ITC) is a technique that is capable of quantifying the stoichiometry, equilibrium constants and thermodynamics of molecular binding events. Thus, important information about the interaction of metal ions with biological macromolecules can be obtained with ITC measurements. This review highlights many of the recent studies of metal ions binding to proteins that have used ITC to quantify the thermodynamics of metal-protein interactions.     

A study of the molecular mechanism of DNA interaction with divalent metal ions

The interaction of DNA with divalent metal ions: Ba2+, Mg2+, Mn2+, Ni2+, Cu2+ in solutions at different ionic strengths mu was investigated. The combination of following methods: flow birefringence, viscometry, UV-spectroscopy and circular dichroism made possible to follow the state of the secondary and tertiary structure of the DNA molecule during its interaction with ions. The presence of divalent ions in solution affects the hydrodynamic properties of DNA only at low mu. At high mu the difference in the action of mono- and divalent ions disappears. The persistence length of DNA does not change during the experiment. It is shown that the Mg2+ and Ba2+ ions interact only with phosphate groups of DNA but Mn2+, Ni2+, Cu2+ ions interact also with the nitrogen bases of the macromolecule.     

DNA interaction with Mn2+ ions at elevated temperatures: VCD evidence of DNA aggregation

Interaction of natural calf thymus DNA with Mn(2+) ions was studied at room temperature and at elevated temperatures in the range from 23 degrees C to 94 degrees C by means of IR absorption and vibrational circular dichroism (VCD) spectroscopy. The Mn(2+) concentration was varied between 0 and 1.3M (0 and 10 [Mn]/[P]). The secondary structure of DNA remained in the frame of the B-form family in the whole ion concentration range at room temperature. No significant DNA denaturation was revealed at room temperature even at the highest concentration of metal ions studied. However at elevated temperatures, DNA denaturation and a significant decrease of the melting temperature of DNA connected with a decrease of the stability of DNA induced by Mn(2+) ions occurred. VCD demonstrated sensitivity to DNA condensation and aggregation as well as an ability to distinguish between these two processes. No condensation or aggregation of DNA was observed at room temperature at any of the metal ion concentrations studied. DNA condensation was revealed in a very narrow range of experimental conditions at around 2.4 [Mn]/[P] and about 55 degrees C. DNA aggregation was observed in the presence of Mn(2+) ions at elevated temperatures during or after denaturation. VCD spectroscopy turned out to be useful for studying DNA condensation and aggregation due to its ability to distinguish between these two processes, and for providing information about DNA secondary structure in a condensed or aggregated state.     

Interactions of DNA with divalent metal ions. III. Extent of metal binding: Experiment and theory

DNA with Mn²⁺ as the only counterion has been prepared, and the extent of the Mn²⁺ binding was determined under a variety of conditions through measurements of the proton relaxation enhancement of water. The total extent of Mn²⁺ binding per DNA phosphate is found to be 0.43 ± 0.04, independent of the metal ion concentration in the experimental range of 2.8 × 10^?5 to 2.1 × 10^?3M. The predictions of Manning's condensation theory and those obtained from solution of the generalized Poisson-Boltzmann equation regarding the extent of divalent ion binding to polyelectrolytes, in the presence and absence of monovalent counterions, are compared with one another and with the experimental results. Good agreement between the two theoretical approaches is found, with less than 14% variance in the predicted extent of binding over a large range of mono- and divalent ion concentrations. While the predictions of both theoretical approaches generally agree with the experimental results, some discrepancies are noted and their possible origins discussed.     

On the importance of van der Waals interaction in the groove binding of DNA with ligands: restrained molecular dynamics study

Comparison of interaction energy between an oligonucleotide and a DNA-binding ligand in the minor and major groove modes was made by use of restrained molecular dynamics. Distortion in DNA was found for the major groove mode whereas less significant changes for both ligand and DNA were detected for the minor groove binding after molecular dynamics simulation. The conformation of the ligand obtained from the major groove mode resembles that computed with the ligand soaked in water. The van der Waals contact energy was found to be as significant as electrostatic energy and more important for difference in binding energy between these two binding modes. The importance of van der Waals force in groove binding was supported by computations on the complex formed by the repressor peptide fragment from the bacteriophage 434 and its operator oligonucleotide.     

Acrylonitrile interaction with testicular DNA in rats

In the present study we report the in vivo interaction of acrylonitrile (VCN) with testicular tissue in rats. Covalent binding of radioactivity to testicular tissue DNA was examined for a period of 72 hr after a single oral dose (46.5 mg/kg) of [2, 3-¹⁴C] VCN. Maximal covalent binding was observed at 0.5 hr (8.9 μmol VCN equivalent/mol nucleotide). Binding decreased gradually thereafter but was still detected (2.5 μmol VCN equivalent/mol nucleotide) at 72 hr following VCN administration. Further, we examined the effects of VCN on DNA synthesis and repair in the testes of rats following a single oral dose (46.5 mg/kg) of VCN to clarify the impact of the covalent binding observed on the testicular genetic material. A significant decrease in DNA synthesis (80% of control) was observed at 0.5 hr after treatment. At 24 hr following acrylonitrile administration, testicular DNA synthesis was severely inhibited (38% of control). Testicular DNA repair was increased 1.5-fold at 0.5 hr and more than 3.3-fold at 24 hr following treatment with VCN. These results suggest that VCN can act as a multipotent genotoxic agent by alkylating DNA in testicular tissue and may affect the male reproductive function by interfering with testicular DNA synthesis and repair processes.     

Towards a model of non-equilibrium binding of metal ions in biological systems

We have used a systems biology approach to address the hitherto insoluble problem of the quantitative analysis of non-equilibrium binding of aqueous metal ions by competitive ligands in heterogeneous media. To-date, the relative proportions of different metal complexes in aqueous media has only been modelled at chemical equilibrium and there are no quantitative analyses of the approach to equilibrium. While these models have improved our understanding of how metals are used in biological systems they cannot account for the influence of kinetic factors in metal binding, transport and fate. Here we have modelled the binding of aluminium, Al(III), in blood serum by the iron transport protein transferrin (Tf) as it is widely accepted that the biological fate of this non-essential metal is not adequately described by experiments, invitro and insilico, which have consistently demonstrated that at equilibrium 90% of serum Al(III) is bound by Tf. We have coined this paradox ‘the blood-aluminium problem’ and herein applied a systems biology approach which utilised well-found assumptions to pare away the complexities of the problem such that it was defined by a comparatively simple set of computational rules and, importantly, its solution assumed significant predictive capabilities. Here we show that our novel computational model successfully described the binding of Al(III) by Tf both at equilibrium and as equilibrium for Al_Tf was approached. The model predicted significant non-equilibrium binding of Al by ligands in competition with Tf and, thereby, provided an explanation of why the distribution of Al(III) in the body cannot be adequately described by its binding and transport by Tf alone. Generically the model highlighted the significance of kinetic in addition to thermodynamic constraints in defining the fate of metal ions in biological systems.     

Preliminary dielectric measurement and analysis protocol for determining the melting temperature and binding energy of short sequences of DNA in solution

Measurement of the real dielectric constant of bulk buffer solutions containing short sequences of DNA as a function of temperature through the DNA melting or denaturiztion transition can be used to determine melting temperatures, T(m), and to estimate the binding energy of the complimentary strands. We describe a preliminary dielectric measurement and analysis protocol to determine these parameters and its application to two known short sequences. The relative real dielectric constant for the bulk solutions was determined over the frequency range of 50 Hz-20 kHz and temperature range of <40-65 degrees C. The measurements were performed on dilute solutions and utilized low electric field strengths. Based on fits to the data by modified sigmoid functions, the melting temperatures, width of transition, and binding energy for the two sequences in solution were estimated. It was observed that the order of the transition appeared to be second order. The results were then compared against predictions of a number of models from the literature that provide theoretical estimates for the melting temperatures of known short sequences of DNA.     

Thymol and carvacrol binding to DNA: model for drug-DNA interaction

Thymol and carvacrol can bind to major and minor grooves of B-DNA. The aim of this study was to examine the interaction of calf thymus DNA with thymol and carvacrol in aqueous solution and physiological pH with thymol/DNA and carvacrol/DNA (phosphate) molar ratios of 1/20, 1/10, 1/5, and 1/1. Fourier transform infrared and UV-visible difference spectroscopy were used to determine the thymol and carvacrol binding mode, binding constant, sequence selectivity, DNA secondary structure, and structural variations of thymol/DNA and carvacrol/DNA complexes in aqueous solution. Spectroscopic evidence showed that the thymol and carvacrol interaction occurred mainly through H-bonding of the thymol and carvacrol OH group to the guanine N7, cytosine N3, and backbone phosphate group with overall binding constant of K(thymol-DNA) = 2.43 x 10(3) M(-1), K(carvacrol-DNA) = 1.55 x 10(3) M(-1). In thymol and carvacrol-DNA complexes, DNA remains in the B-family structure.     

Quantum dot binding to DNA: Single-molecule imaging with atomic force microscopy

The interaction between nanoparticles (NPs) and DNA is of significance for both application and implication research of NPs. In this study, a single-molecule imaging technique based on atomic force microscopy (AFM) was employed to probe the NP-DNA interactions with quantum dots (QDs) as model NPs. Reproducible high-quality images of single DNA molecules in air and in liquids were acquired on mica by optimizing sample preparation conditions. Furthermore, the binding of QDs to DNA was explored using AFM. The DNA concentration was found to be a key factor influencing AFM imaging quality. In air and liquids, the optimal DNA concentration for imaging DNA molecules was approximately 2.5 and 0.25 μg/mL, and that for imaging DNA binding with QDs was 0.5 and 0.25 μg/mL, respectively. In the presence of QDs, the DNA conformation was altered with the formation of DNA condensates. Finally, the fine conformation of QD-DNA binding sites was examined to analyze the binding mechanisms. This work will benefit investigations of NP-DNA interactions and the understanding of the structure of NP-DNA bioconjugates. See accompanying article by Wang DOI: 10.1002/biot.201200309     

Thermostable DNA polymerase from Thermus thermophilus B35: influence of divalent metal ions on the interaction with deoxynucleoside triphosphates

The interaction of DNA polymerase from Thermus thermophilus B35 (Tte-pol) with deoxynucleoside triphosphates in the presence of different divalent metal ions has been studied. DNA synthesis and competitive inhibition of the polymerase reaction by non-complementary dNTPs are described with corresponding kinetic schemes. The co-factor properties of some metals (Mg2+, Mn2+, Co2+, Ni2+, Cu2+, Ca2+, Cd2+, and Zn2+) were investigated, and their activating concentration ranges were determined. It was found that kcat values are significantly decreased and Km values slowly decrease when Mn2+ displaces Mg2+. The value of Kd for DNA template-primer is Me2+-independent, whereas Kd values for non-complementary dNTPs decrease in the presence of Mn2+. Tte-pol processivity but not DNA synthesis efficiency is Me2+-type independent.     

Complexation of a four-strand tetraalcohol with labile metal ions probed by electrospray mass spectrometry

The saturated, stereodefined tetraalcohol 2,3,5,6-endo,endo,endo,endo-tetrakis(hydroxymethyl)bicyclo[2.2.1]heptane (tetol, L¹) and the simple alcohol butane-1,3-diol (L²) form complexes with alkali metal ions (lithium, sodium, potassium, rubidium and caesium), alkali earth cations (magnesium, calcium, strontium and barium) and Ga(III) and Ce(IV) in aqueous solution, characterised by electrospray ionisation mass spectrometry (ESMS). Metal ion exchange between the Li⁺ complex of L¹ and the other metal ions is rapid, with a range of M(L¹)_n ^m+ species detected, in addition to solvated species. With the alkal metal ions, M(L¹)⁺ and M(L¹)₂ ⁺ are dominant, although speciation varies with metal ion size. For the alkaline earth ions, a range of complex ions up to n=8 are observed, although n=1-3 dominate. A preference for M(L¹)₂ ²⁺ with Mg²⁺ versus M(L¹)₃ ²⁺ with Ca²⁺ may again relate to a larger ion size. For the higher-charged Ga(III) and Ce(IV), hydroxo species M(OH)(L¹)_n ^(m−1)+ are dominant reflecting bulk solution behaviour, which the ESMS studies appear to map generally.     

Structure and binding of Mg(II) ions and di-metal bridge complexes with biological phosphates and phosphoranes

Divalent Mg²⁺ ions often serve as cofactors in enzyme or ribozyme-catalyzed phosphoryl transfer reactions. In this work, the interaction of Mg²⁺ ions and di-metal bridge complexes with phosphates, phosphoranes, and other biological ligands relevant to RNA catalysis are characterized with density functional methods. The effect of bulk solvent is treated with two continuum solvation methods (PCM and COSMO) for comparison. The relative binding affinity for different biological ligands to Mg²⁺ are quantified in different protonation states. The structure and stability of the single-metal and di-metal complexes are characterized, and the changes in phosphate and phosphorane geometry induced by metal ion binding are discussed. Di-metal bridge complexes are a ubiquitous motif and the key factors governing their electrostatic stabilization are outlined. The results presented here provide quantitative characterization of metal ion binding to ligands of importance to RNA catalysis, and lay the groundwork for design of new generation quantum models that can be applied to the full biological enzymatic systems.Electronic Supplementary Material Supplementary material is available in the online version of this article at http://dx.doi.org/10.1007/s00775-004-0583-7An erratum to this article can be found at     

Daunomycin interaction with DNA: Microcalorimetric studies of the thermodynamics and binding mechanism

Nucleic acids are an important target for many therapeutics. Small molecules that bind to nucleic acids are important in many aspects of medicines, particularly in cancer chemotherapy. In recent years, many studies have utilized polynucleic acids with various sequences to demonstrate the binding mechanism of daunomycin, a potent anticancer drug. This study describes that isothermal titration calorimetry is a useful tool for studying the fundamental binding mechanism systemically. The results suggest that the binding free energy is more favorable when the temperature is increased. The binding entropy contributes to this effect. Furthermore, the amine group on daunomycin contributes electrostatic interaction that induces the binding process. In addition, enthalpy-entropy compensation is also exhibited in the daunomycin-DNA binding mechanism. This study used an easy, convenient method of performing a systemic study in a recognition system. The results from this study provide additional information about microscopic mechanisms for molecular design and molecular recognition.