Magnesium Binding Research Articles

Magnesium binding by terrestrial humic acids

Environmental contextThe behaviour of magnesium, which is an essential element for all living organisms, in terrestrial environments is influenced by natural organic matter. This study shows that magnesium binding by terrestrial humic acids exhibits a pronounced ionic strength-dependence indicating a strong preference for electrostatic binding to humic acids. This interaction is expected to influence the mobility of humic substances and their associated trace elements. AbstractMagnesium binding by three terrestrial humic acids was investigated at pH 8 and 25 °C as a function of Mg2+ activity and ionic strength using NaCl as the background electrolyte. The Mg2+ activity in solution was directly measured with an Mg2+-selective electrode in the titration experiments. In addition, coagulation experiments using Ca2+ and Mg2+ as the coagulants were carried out at pH 8. For the titration data, the NICA–Donnan model was used to quantitatively describe Mg2+ binding to the humic acids considering electrostatic and specific Mg2+ binding. Mg2+ binding to humic acids was found to be strongly affected by ionic strength variations indicating that Mg2+ binding largely arose from electrostatic (nonspecific) interactions with negatively charged functional groups of the humic acids. Data modelling suggested that the relative contribution of specific binding increased with decreasing Mg2+ activity and was related to functional groups with low proton affinities. For all three humic acids studied, the fitted Mg2+ affinity constants for specific binding were lower than the respective Ca2+ affinities. Corresponding to the observed differences in cation binding and the known differences in ion hydration, Ca2+ was observed to be the stronger coagulant as compared with Mg2+. The results suggest that Mg2+ may influence the mobility of trace elements that are strongly bound to humic acids such as mercury, although Mg2+ is not expected to directly compete with strongly sorbing elements for specific binding.

Effect of additives on hydration and hardening of magnesia compositions

The article is devoted to the investigation of the influence of technological factors on the hydration and hardening of magnesia compositions. The objective – is investigation magnesia compositions with different additives. Factors which impact activity of magnesium oxide in compositions of different structure are investigated. Influence of liquid density on hardening of magnesium bindings is defined. Processes of hydration and hardening of magnesium bindings with participation of minerals – silicates are investigated. It is revealed that the addition of semi-aquatic calcium sulfate contributes to the hardening of magnesia binders. Defined effect of concentration calcium sulfate hemihydrate to the hardening of caustic magnesite. Here are proposed structures of sulphomagnesium compositions containing technogenic components. It revealed a beneficial effect on the hardening of ferrous component of the mixed magnesia binder. Composition of hydration products of magnesium binding with participation of ferriferous minerals is presented. Transformations of phases at hydration of magnesium binding are revealed. Influence of structure of bindings on transformations of hydrates is established. Results of research of magnesia bindings of long hardening are given. It is shown that the durability of stone of bindings is provided with dense structure of hydrates. In researches are used X – Ray and thermal methods, electronic microscopy.

Relative association of Rubisco with manganese and magnesium as a regulatory mechanism in plants

Rubisco, the enzyme that constitutes as much as half of the protein in a leaf, initiates either the photorespiratory pathway that supplies reductant for the assimilation of nitrate into amino acids or the C3 carbon fixation pathway that generates carbohydrates. The relative rates of these two pathways depend both on the relative extent to which O2 and CO2 occupies the active site of Rubisco and on whether manganese or magnesium is bound to the enzyme. This study quantified the activities of manganese and magnesium in isolated tobacco chloroplasts and the thermodynamics of binding of these metals to Rubisco purified from tobacco or a bacterium. In tobacco chloroplasts, manganese was less active than magnesium, but Rubisco purified from tobacco had a higher affinity for manganese. The activity of each metal in the chloroplast was similar in magnitude to the affinity of tobacco Rubisco for each. This indicates that, in tobacco chloroplasts, Rubisco associates almost equally with both metals and rapidly exchanges one metal for the other. Binding of magnesium was similar in Rubisco from tobacco and a bacterium, whereas binding of manganese differed greatly between the Rubisco from these species. Moreover, the ratio of leaf manganese to magnesium in C3 plants increased as atmospheric CO2 increased. These results suggest that Rubisco has evolved to improve the energy transfers between photorespiration and nitrate assimilation and that plants regulate manganese and magnesium activities in the chloroplast to mitigate detrimental changes in their nitrogen/carbon balance as atmospheric CO2 varies.

Cell differentiation defines acute and chronic infection cell types in Staphylococcus aureus.

A central question to biology is how pathogenic bacteria initiate acute or chronic infections. Here we describe a genetic program for cell-fate decision in the opportunistic human pathogen Staphylococcus aureus, which generates the phenotypic bifurcation of the cells into two genetically identical but different cell types during the course of an infection. Whereas one cell type promotes the formation of biofilms that contribute to chronic infections, the second type is planktonic and produces the toxins that contribute to acute bacteremia. We identified a bimodal switch in the agr quorum sensing system that antagonistically regulates the differentiation of these two physiologically distinct cell types. We found that extracellular signals affect the behavior of the agr bimodal switch and modify the size of the specialized subpopulations in specific colonization niches. For instance, magnesium-enriched colonization niches causes magnesium binding to S. aureusteichoic acids and increases bacterial cell wall rigidity. This signal triggers a genetic program that ultimately downregulates the agr bimodal switch. Colonization niches with different magnesium concentrations influence the bimodal system activity, which defines a distinct ratio between these subpopulations; this in turn leads to distinct infection outcomes in vitro and in an in vivo murine infection model. Cell differentiation generates physiological heterogeneity in clonal bacterial infections and helps to determine the distinct infection types.

Phenotypic and molecular characterisation of CDK13-related congenital heart defects, dysmorphic facial features and intellectual developmental disorders

BackgroundDe novo missense variants in CDK13 have been described as the cause of syndromic congenital heart defects in seven individuals ascertained from a large congenital cardiovascular malformations cohort. We aimed to further define the phenotypic and molecular spectrum of this newly described disorder.MethodsTo minimise ascertainment bias, we recruited nine additional individuals with CDK13 pathogenic variants from clinical and research exome laboratory sequencing cohorts. Each individual underwent dysmorphology exam and comprehensive medical history review.ResultsWe demonstrate greater than expected phenotypic heterogeneity, including 33% (3/9) of individuals without structural heart disease on echocardiogram. There was a high penetrance for a unique constellation of facial dysmorphism and global developmental delay, as well as less frequently seen renal and sacral anomalies. Two individuals had novel CDK13 variants (p.Asn842Asp, p.Lys734Glu), while the remaining seven unrelated individuals had a recurrent, previously published p.Asn842Ser variant. Summary of all variants published to date demonstrates apparent restriction of pathogenic variants to the protein kinase domain with clustering in the ATP and magnesium binding sites.ConclusionsHere we provide detailed phenotypic and molecular characterisation of individuals with pathogenic variants in CDK13 and propose management guidelines based upon the estimated prevalence of anomalies identified.

Computational study of fluoroquinolone binding to Mg(H2O)N2+ and its applicability to future drug design

Fluoroquinolones are an important therapeutic class in the targeting of new and resistant bacterial infections. Fluoroquinolones bind to bacterial type II topoisomerase via a water-Mg2+ bridge. However, binding to magnesium-containing molecules outside of the target cells increases the minimum inhibitory concentration (MIC) and promotes drug resistance. As a result, fluoroquinolones are counter-indicated with magnesium and multivalent metal cation containing drugs, such as antacids. The antibiotic efficacy of fluoroquinolones has also been shown to be pH dependent, as we show the effect of protonation state on magnesium binding. This work presents a systematic computational study of fluoroquinolones' magnesium-binding properties. We use B3LYP density functional theory and triple-zeta basis sets, to evaluate Mg(H2O)N2+ binding affinities. Complexation is predicted to be thermodynamically favorable at neutral and basic compared to acidic pH. The calculated complexation energies broadly capture experimental binding affinities, suggesting this is a valid approach for designing new fluoroquinolones with a target magnesium binding affinity. We also investigate the effect of chemical substitution at the carboxylic acid to help in the identification of potential new antibiotics based on the fluoroquinolone pharmacophore.

Predicting effects on oxaliplatin clearance: in vitro, kinetic and clinical studies of calcium- and magnesium-mediated oxaliplatin degradation

This study evaluated the impact of calcium and magnesium on the in vitro degradation and in vivo clearance of oxaliplatin. Intact oxaliplatin and Pt(DACH)Cl2 were measured in incubation solutions by HPLC-UV. A clinical study determined changes in plasma concentrations of calcium and magnesium in cancer patients and their impact on oxaliplatin clearance. Kinetic analyses modelled oxaliplatin degradation reactions in vitro and contributions to oxaliplatin clearance in vivo. Calcium and magnesium accelerated oxaliplatin degradation to Pt(DACH)Cl2 in chloride-containing solutions in vitro. Kinetic models based on calcium and magnesium binding to a monochloro-monooxalato ring-opened anionic oxaliplatin intermediate fitted the in vitro degradation time-course data. In cancer patients, calcium and magnesium plasma concentrations varied and were increased by giving calcium gluconate and magnesium sulfate infusions, but did not alter or correlate with oxaliplatin clearance. The intrinsic in vitro clearance of oxaliplatin attributed to chloride-, calcium- and magnesium-mediated degradation predicted contributions of <2.5% to the total in vivo clearance of oxaliplatin. In conclusion, calcium and magnesium accelerate the in vitro degradation of oxaliplatin by binding to a monochloro-monooxalato ring-opened anionic intermediate. Kinetic analysis of in vitro oxaliplatin stability data can be used for in vitro prediction of potential effects on oxaliplatin clearance in vivo.

The structural switch of nucleotide-free kinesin

Kinesin-1 is an ATP-dependent motor protein that moves towards microtubules (+)-ends. Whereas structures of isolated ADP-kinesin and of complexes with tubulin of apo-kinesin and of ATP-like-kinesin are available, structural data on apo-kinesin-1 in the absence of tubulin are still missing, leaving the role of nucleotide release in the structural cycle unsettled. Here, we identified mutations in the kinesin nucleotide-binding P-loop motif that interfere with ADP binding. These mutations destabilize the P-loop (T87A mutant) or magnesium binding (T92V), highlighting a dual mechanism for nucleotide release. The structures of these mutants in their apo form are either isomorphous to ADP-kinesin-1 or to tubulin-bound apo-kinesin-1. Remarkably, both structures are also obtained from the nucleotide-depleted wild-type protein. Our results lead to a model in which, when detached from microtubules, apo-kinesin possibly occupies the two conformations we characterized, whereas, upon microtubule binding, ADP-kinesin converts to the tubulin-bound apo-kinesin conformation and releases ADP. This conformation is primed to bind ATP and, therefore, to run through the natural nucleotide cycle of kinesin-1.

Computational Assessment of Potassium and Magnesium Ion Binding to a Buried Pocket in GTPase-Associating Center RNA.

An experimentally well-studied model of RNA tertiary structures is a 58mer rRNA fragment, known as GTPase-associating center (GAC) RNA, in which a highly negative pocket walled by phosphate oxygen atoms is stabilized by a chelated cation. Although such deep pockets with more than one direct phosphate to ion chelation site normally include magnesium, as shown in one GAC crystal structure, another GAC crystal structure and solution experiments suggest potassium at this site. Both crystal structures also depict two magnesium ions directly bound to the phosphate groups comprising this controversial pocket. Here, we used classical molecular dynamics simulations as well as umbrella sampling to investigate the possibility of binding of potassium versus magnesium inside the pocket and to better characterize the chelation of one of the binding magnesium ions outside the pocket. The results support the preference of the pocket to accommodate potassium rather than magnesium and suggest that one of the closely binding magnesium ions can only bind at high magnesium concentrations, such as might be present during crystallization. This work illustrates the complementary utility of molecular modeling approaches with atomic-level detail in resolving discrepancies between conflicting experimental results.

Functional roles of magnesium binding to extracellular signal-regulated kinase 2 explored by molecular dynamics simulations and principal component analysis

Molecular dynamics (MD) simulations coupled with principal component (PC) analysis were carried out to study functional roles of Mg2+ binding to extracellular signal-regulated kinase 2 (ERK2). The results suggest that Mg2+ binding heavily decreases eigenvalue of the first principal component and totally inhibits motion strength of ERK2, which favors stabilization of ERK2 structure. Binding free energy predictions indicate that Mg2+ binding produces an important effect on binding ability of adenosine triphosphate (ATP) to ERK2 and strengthens the ATP binding. The calculations of residue-based free energy decomposition show that lack of Mg2+ weakens interactions between the hydrophobic rings of ATP and five residues I29, V37, A50, L105, and L154. Hydrogen bond analyses also prove that Mg2+ binding increases occupancies of hydrogen bonds formed between ATP and residues K52, Q103, D104, and M106. We expect that this study can provide a significant theoretical hint for designs of anticancer drugs targeting ERK2.

Modulation of Conformational Equilibria in the S-Adenosylmethionine (SAM) II Riboswitch by SAM, Mg(2+), and Trimethylamine N-Oxide.

The dependence of the conformation of the S-adenosylmethionine (SAM) II riboswitch on the concentration of added Mg(2+) ions and SAM, individually and in mixtures, was monitored by circular dichroism (CD) spectroscopy and by measurement of the diffusion coefficient. The results are analyzed in the context of two complementary quantitative models, both of which are consistent with a single underlying physical model. Magnesium binding sites in the open state have an affinity on average higher than the affinity of those in the compact state, but formation of the compact state is accompanied by an increase in the number of binding sites. Consequently, at low Mg(2+) concentrations, Mg(2+) binds preferentially to the open state, favoring its formation, but at high concentrations, Mg(2+) binds preferentially to the compact state. The affinity of the riboswitch for SAM increases drastically with an increased level of binding of Mg(2+) to the compact pseudoknot conformation. The effect of increasing concentrations of trimethylamine N-oxide (TMAO), a well-studied molecular crowding agent, on the conformation of the riboswitch and its affinity for SAM were also monitored by CD spectroscopy and measurement of diffusion. In the absence of added Mg(2+), high concentrations of TMAO were found to induce a conformational change compatible with the formation of the pseudoknot form but have only a small effect on the affinity of the RNA for SAM.

Proteomic analysis and immunodetection of antigens from early developmental stages of Trichinella spiralis

Trichinella spiralis is an ubiquitous parasitic nematode that lives in muscle tissue of many hosts and causes trichinellosis in humans. Numerous efforts have been directed at specific detection of this infection and strategies for its control. TSL-1 and other antigens, mainly from muscle larvae (ML), have been used to induce partial protection in rodents. An improvement in protective immunity may be achieved by using antigens from other parasite stages. Further, identification of other parasite antigens may provide insights into their role in the host-parasite interaction. In this study, T. spiralis antigens from early developmental parasite stages, namely ML and pre-adult (PA) obtained at 6h, 18h and 30h post-infection, were identified by proteomic and mass spectrometry analyses. Our findings showed a differential expression of several proteins with molecular weights in the range of 13–224kDa and pI range of 4.54–9.89. Bioinformatic analyses revealed a wide diversity of functions in the identified proteins, which include structural, antioxidant, actin binding, peptidyl prolyl cis-trans isomerase, motor, hydrolase, ATP binding, magnesium and calcium binding, isomerase and translation elongation factor. This, together with the differential recognition of antigens from these parasite stages by antibodies present in intestinal fluid, in supernatants from intestinal explants, and in serum samples from mice infected with T. spiralis or re-infected with this parasite, provides information that may lead to alternatives in the design of vaccines against this parasite or for modulation of immune responses.

Magnesium and manganese binding sites on proteins have the same predominant motif of secondary structure

Manganese ion (Mn2+) can substitute magnesium ion (Mg2+) in active sites of numerous enzymes. Binding sites for these two ions have been studied in two sets of protein 3D structures from the Protein Data Bank with the homology level lower than 25%. The structural motif “beta strand – binder – random coil” is predominant in both Mn2+ and Mg2+ coordination spheres, especially in functionally relevant ones. That predominant motif works as an active binder of those divalent cations which can then attract additional ligands, such as different phosphate-containing compounds. In contrast, such Mg2+ and Mn2+ binding motif as “GK(T/S)T” being the N-terminal part of alpha helices works as an active binder of phosphates which can then attract divalent cations. There are few differences between Mg2+ and Mn2+ coordination spheres responsible of the cation specificity. His residues are underrepresented in certain positions around Asp and Glu residues involved in Mg2+ coordination, while they are overrepresented in certain positions around Asp and Glu residues coordinating Mn2+. The random coil region in the “beta strand – random coil – alpha helix” motif for Mg2+ binding is usually shorter than that in the same motif for Mn2+ coordination. This feature is associated with the lower number of binding amino acids (and lower levels of usage of such “major” binders as Asp and Glu) for Mg2+ (which is a hard Lewis acid) in comparison with those for Mn2+ (an intermediate Lewis acid).

Dynamic Network Analysis of Dream Protein Dimer Interface

Here we present the use of molecular dynamics network analysis for the study of oligomerization interface of the downstream regulatory element antagonist modulator (DREAM) protein. This calcium binding protein has been shown to regulate numerous processes in neuronal cells in a calcium-dependent manner. Calcium binding to DREAM is known to induce protein structural changes that lead to dimerization and dissociation from the DRE promoter of c-fos, c-jun and dynorphin genes. The association of calcium to DREAM is also known to induce protein dimerization but whether ligand-induced association is regulated by tertiary of quaternary structural changes is not known. Using theoretical and experimental biophysics we investigated the interface of DREAM and identified a leucine-rich region at the exiting helix of EF-hand 2 as well as the dynamic loop connecting EF-hand 3 and EF-hand 4 as being important for dimerization. Bioinformatics analysis show that these regions are conserved among dimeric neuronal calcium sensors but not in NCS-1, which forms monomers is solution. To confirm the role of these regions in dimerization we constructed a DREAM-NCS chimera where these regions were mutated to those observes in NCS-1. We show that these mutations induce the formation of monomers as well as tryptophan transitions on DREAM upon calcium binding and magnesium binding which are similar to those observed for wild-type NCS-1. These findings support an approach where molecular dynamics can be used to identify and disrupt dimeric interfaces on neuronal calcium sensors, and could potentially be translated to other proteins.

Molecular Dynamics Study of Divalent Ion Coordination in EF Hand Proteins

Calcium-binding proteins play an important role in cellular functions and regulation. Two such proteins are the S100A1 protein and the beta-parvalbumin (PV) protein, both of which contain metal-binding sites called EF-hands that consist of helix-loop-helix regions. Both of these proteins selectively bind calcium ions despite significantly higher (millimolar) intracellular concentrations of other cations such as magnesium. While experimental methods such as X-ray crystallography and NMR provide static snapshots that suggest the structural basis of binding selectivity, additional thermodynamic information could be gleaned from the proteins’ dynamic conformations in equilibrium. While it is known that calcium selectivity over magnesium is dependent on the carbonyl coordination in the EF hands, we sought to understand how the dynamics of these carbonyls, as well as global protein motions, might contribute to the energetics of selectivity. To explore how calcium binding drives global motions, we ran 100 ns molecular dynamics simulations of the S100A1 proteins in the unsaturated (apo), fully saturated (holo), and half-saturated states (one of two bound calciums per subunit). Our simulations indicate that the half-saturated state missing half the calcium ions is unstable and starts to revert to the closed form. To understand the molecular basis of calcium versus magnesium, we ran 100 ns simulations on the wild-type PV plus three mutants with mutations in the EF hands (S55D, E62D, S55D/E62D) that have been experimentally shown to impact the calcium and the magnesium binding affinities. We observe significant differences in the structures of the EF hands and the coordinating carbonyls in the presence of magnesium or calcium for the four proteins. These studies explain how the chelating oxygens, the EF hand structure, and the overall stability of the protein influence the calcium binding affinity and selectivity on the molecular scale.

The Role of Parvalbumin, Sarcoplasmatic Reticulum Calcium Pump Rate, Rates of Cross-Bridge Dynamics, and Ryanodine Receptor Calcium Current on Peripheral Muscle Fatigue: A Simulation Study.

A biophysical model of the excitation-contraction pathway, which has previously been validated for slow-twitch and fast-twitch skeletal muscles, is employed to investigate key biophysical processes leading to peripheral muscle fatigue. Special emphasis hereby is on investigating how the model's original parameter sets can be interpolated such that realistic behaviour with respect to contraction time and fatigue progression can be obtained for a continuous distribution of the model's parameters across the muscle units, as found for the functional properties of muscles. The parameters are divided into 5 groups describing (i) the sarcoplasmatic reticulum calcium pump rate, (ii) the cross-bridge dynamics rates, (iii) the ryanodine receptor calcium current, (iv) the rates of binding of magnesium and calcium ions to parvalbumin and corresponding dissociations, and (v) the remaining processes. The simulations reveal that the first two parameter groups are sensitive to contraction time but not fatigue, the third parameter group affects both considered properties, and the fourth parameter group is only sensitive to fatigue progression. Hence, within the scope of the underlying model, further experimental studies should investigate parvalbumin dynamics and the ryanodine receptor calcium current to enhance the understanding of peripheral muscle fatigue.

Selective Inhibition of Mutant Isocitrate Dehydrogenase 1 (IDH1) via Disruption of a Metal Binding Network by an Allosteric Small Molecule

Cancer-associated point mutations in isocitrate dehydrogenase 1 and 2 (IDH1 and IDH2) confer a neomorphic enzymatic activity: the reduction of α-ketoglutarate to d-2-hydroxyglutaric acid, which is proposed to act as an oncogenic metabolite by inducing hypermethylation of histones and DNA. Although selective inhibitors of mutant IDH1 and IDH2 have been identified and are currently under investigation as potential cancer therapeutics, the mechanistic basis for their selectivity is not yet well understood. A high throughput screen for selective inhibitors of IDH1 bearing the oncogenic mutation R132H identified compound 1, a bis-imidazole phenol that inhibits d-2-hydroxyglutaric acid production in cells. We investigated the mode of inhibition of compound 1 and a previously published IDH1 mutant inhibitor with a different chemical scaffold. Steady-state kinetics and biophysical studies show that both of these compounds selectively inhibit mutant IDH1 by binding to an allosteric site and that inhibition is competitive with respect to Mg(2+). A crystal structure of compound 1 complexed with R132H IDH1 indicates that the inhibitor binds at the dimer interface and makes direct contact with a residue involved in binding of the catalytically essential divalent cation. These results show that targeting a divalent cation binding residue can enable selective inhibition of mutant IDH1 and suggest that differences in magnesium binding between wild-type and mutant enzymes may contribute to the inhibitors' selectivity for the mutant enzyme.

ATP and Magnesium Promote Cotton Short-Form Ribulose-1,5-bisphosphate Carboxylase/Oxygenase (Rubisco) Activase Hexamer Formation at Low Micromolar Concentrations

We report a fluorescence correlation spectroscopy (FCS) study of the assembly pathway of the AAA+ protein ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) activase (Rca), a ring-forming ATPase responsible for activation of inhibited Rubisco complexes for biological carbon fixation. A thermodynamic characterization of simultaneously populated oligomeric states appears critical in understanding Rca structure and function. Using cotton β-Rca, we demonstrate that apparent diffusion coefficients vary as a function of concentration, nucleotide, and cation. Using manual fitting procedures, we provide estimates for the equilibrium constants for the stepwise assembly and find that in the presence of ATPγS, the Kd for hexamerization is 10-fold lower than with ADP (∼0.1 vs ∼1 μM). Hexamer fractions peak at 30 μM and dominate at 8-70 μM Rca, where they comprise 60-80% of subunits with ATPγS, compared with just 30-40% with ADP. Dimer fractions peak at 1-4 μM Rca, where they comprise 15-18% with ATPγS and 26-28% with ADP. At 30 μM Rca, large aggregates begin to form that comprise ∼10% of total protein with ATPγS and ∼25% with ADP. FCS data collected on the catalytically impaired WalkerB-D173N variant in the presence of ATP provided strong support for these results. Titration with free magnesium ions lead to the disaggregation of larger complexes in favor of hexameric forms, suggesting that a second magnesium binding site with a Kd value of 1-3 mM mediates critical subunit contacts. We propose that closed-ring toroidal hexameric forms are stabilized by binding of Mg·ATP plus Mg2+, whereas Mg·ADP promotes continuous assembly to supramolecular aggregates such as spirals.

Revised model of calcium and magnesium binding to the bacterial cell wall.

Metals bind to the bacterial cell wall, yet the binding mechanisms and affinity constants are not fully understood. The cell wall of gram positive bacteria is characterized by a thick layer of peptidoglycan and anionic teichoic acids anchored in the cytoplasmic membraneas lipoteichoic acid or covalently bound to the cell wallas wall teichoic acid. The polyphosphate groups of teichoic acid provide one-half of the metal binding sites for calcium and magnesium, whichcontradicts previous reports that calcium binding is 100% dependent on teichoic acid. The remaining binding sites are formed with the carboxyl units of peptidoglycan. In this work we report equilibrium association constants and total metal binding capacities for the interaction of calcium and magnesium ions with the bacterial cell wall. Metal binding is much stronger than previously reported. Curvature of Scatchard plots from the binding data and the resulting two regions of binding affinity suggest the presence of negative cooperative binding, whichmeans that the binding affinity decreases as more ions become bound to the sample. For Ca(2+), Region I has a KA=(1.0±0.2)×10(6)M(-1) and Region II has a KA=(0.075±0.058)×10(6)M(-1). For Mg(2+), KA1=(1.5±0.1)×10(6) and KA2=(0.17±0.10)×10(6). A binding capacity (η) is reported for both regions. However, since binding is still occurring in Region II, the total binding capacity is denoted by η2, which are 0.70±0.04 and 0.67±0.03µmol/mg for Ca(2+) and Mg(2+) respectively. These data contradict the current paradigm of only a single metal affinity value that is constant over a range of concentrations. We also find that measurement of equilibrium binding constants is highly sample dependent. This suggests a role for diffusion of metals through heterogeneous cell wall fragments. As a result, we are able to reconcile many contradictory theories that describe binding affinity and the binding mode of divalent metal cations.

Characterization and Evolutionary Implications of the Triad Asp-Xxx-Glu in Group II Phosphopantetheinyl Transferases

Phosphopantetheinyl transferases (PPTases), which play an essential role in both primary and secondary metabolism, are magnesium binding enzymes. In this study, we characterized the magnesium binding residues of all known group II PPTases by biochemical and evolutionary analysis. Our results suggested that group II PPTases could be classified into two subgroups, two-magnesium-binding-residue-PPTases containing the triad Asp-Xxx-Glu and three-magnesium-binding-residue-PPTases containing the triad Asp-Glu-Glu. Mutations of two three-magnesium-binding-residue-PPTases and one two-magnesium-binding-residue-PPTase indicate that the first and the third residues in the triads are essential to activities; the second residues in the triads are non-essential. Although variations of the second residues in the triad Asp-Xxx-Glu exist throughout the whole phylogenetic tree, the second residues are conserved in animals, plants, algae, and most prokaryotes, respectively. Evolutionary analysis suggests that: the animal group II PPTases may originate from one common ancestor; the plant two-magnesium-binding-residue-PPTases may originate from one common ancestor; the plant three-magnesium-binding-residue-PPTases may derive from horizontal gene transfer from prokaryotes.