Magnesium Binding Research Articles

Quantum and All-Atom Molecular Dynamics Simulations of Protonation and Divalent Ion Binding to Phosphatidylinositol 4,5-Bisphosphate (PIP2)

Molecular dynamics calculations have been used to determine the structure of phosphatidylinositol 4,5 bisphosphate (PIP2) at the quantum level and to quantify the propensity for PIP2 to bind two physiologically relevant divalent cations, Mg(2+) and Ca(2+). We performed a geometry optimization at the Hartree-Fock 6-31+G(d) level of theory in vacuum and with a polarized continuum dielectric to determine the conformation of the phospholipid headgroup in the presence of water and its partial charge distribution. The angle between the headgroup and the acyl chains is nearly perpendicular, suggesting that in the absence of other interactions the inositol ring would lie flat along the cytoplasmic surface of the plasma membrane. Next, we employed hybrid quantum mechanics/molecular mechanics (QM/MM) simulations to investigate the protonation state of PIP2 and its interactions with magnesium or calcium. We test the hypothesis suggested by prior experiments that binding of magnesium to PIP2 is mediated by a water molecule that is absent when calcium binds. These results may explain the selective ability of calcium to induce the formation of PIP2 clusters and phase separation from other lipids.

Hexaamminecobalt(III) – Probing Metal Ion Binding Sites in Nucleic Acids by NMR Spectroscopy

AbstractHexaamminecobalt(III), an octahedral, inert metal ion complex, has lately gained increasing attention of structural biologists and bioinorganic chemists due to its use in structure determination of nucleic acids. This complex mimics outer‐sphere binding events of the physiologically relevant magnesium(II)hexaaqua ion; hexaamminecobalt(III) often finds usage either in NMR spectroscopy, where cross‐peaks between the complex ammines and the nucleic acid protons near the binding site are observed, in X‐ray spectroscopy as heavy metal derivative for phasing, or in other techniques. In this review, we discuss the basic hexaamminecobalt(III) binding modes and give an overview on the most recent findings on [Co(NH3)6]Cl3–nucleic acid complexes. The various techniques that are applied in combination with this complex are mentioned and briefly summarized. Special attention is given to the application of [Co(NH3)6]Cl3 in nuclear magnetic resonance spectroscopy of nucleic acids, where it is used to reveal potential outer‐sphere magnesium binding sites.

Regulation of mismatch repair protein MutSα functions by its Walker A and Walker B motifs

The mismatch recognition protein MutSα is an MSH2‐MSH6 heterodimer. Mismatch binding by MutSα triggers a series of downstream mismatch repair (MMR) reactions, including interacting and communicating with other MMR proteins by sliding along the DNA helixes. These MutSα biochemical functions rely on its ATP/ADP binding and ATPase activities. Like many ATPases, both MSH2 and MSH6 subunits contain a Walker A motif and a Walker B motif. However, how these Walker motifs contribute to the MutSα functions is not fully understood. To address this issue, a series of MutSα mutants that contain a lysine (K) to alanine substitution in Walker A motif and a glutamate (E) to K substitution in Walker B motif of either MSH2 or MSH6, or both were analyzed for their MMR activities. We show here that MSH2 mutations in Walker A or B motif block MutSα ATP hydrolysis, suggesting that the MSH2 Walker motifs regulate MutSα ATPase activity. Interestingly, the E‐K substitutions in the Walker B motif produce a mutant MutSα that, instead of sliding off, undergoes direct dissociation from a mismatch‐containing DNA molecule regardless of the presence of magnesium. This indicates that the E residue in the Walker B motif of either MSH2 or MSH6 is required for magnesium binding, and is indispensable for MutSα conformational change to form a sliding clamp. Our work provides new insights into how the Walker motifs contribute to MutSα activities in MMR.

Energetics of the Escherichia coli DnaT Protein Trimerization Reaction

Thermodynamic and structural characteristics of the Escherichia coli DnaT protein trimerization reaction have been quantitatively examined using fluorescence anisotropy and analytical ultracentrifugation methods. Binding of magnesium to the DnaT monomers regulates the intrinsic affinity of the DnaT trimerization reaction. Comparison between the DnaT trimer and the isolated N-terminal core domain suggests that magnesium binds to the N-terminal domain but does not associate with the C-terminal region of the protein. The magnesium binding process is complex and involves approximately three Mg(2+) cations per protein monomer. The observed effect seems to be specific for Mg(2+). In the examined salt concentration range, monovalent cations and anions do not affect the trimer assembly process. However, magnesium affects neither the cooperativity of the trimerization reaction nor the GnHCl-induced trimer dissociation, strongly indicating that Mg(2+) indirectly stabilizes the trimer through the induced changes in the monomer structures. Nevertheless, formation of the trimer also involves specific conformational changes of the monomers, which are independent of the presence of magnesium. Binding of Mg(2+) cations dramatically changes the thermodynamic functions of the DnaT trimerization, transforming the reaction from a temperature-dependent to temperature-independent process. Highly cooperative dissociation of the trimer by GnHCl indicates that both interacting sites of the monomer, located on the N-terminal core domain and formed by the small C-terminal region, are intimately integrated with the entire protein structure. In the intact protein, the C-terminal region most probably interacts with the corresponding binding site on the N-terminal domain of the monomer. Functional implications of these findings are discussed.

Quantum and All-Atom Molecular Dynamics Simulations of Protonation and Divalent Ion Binding to Phosphatidylinositol 4,5-Bisphosphate (PIP2)

Molecular dynamics calculations have been used to determine the structure of phosphatidylinositol 4,5 bisphosphate (PIP2) at the quantum level and to quantify the propensity for PIP2 to bind two physiologically relevant divalent cations, Mg(2+) and Ca(2+). We performed a geometry optimization at the Hartree-Fock 6-31+G(d) level of theory in vacuum and with a polarized continuum dielectric to determine the conformation of the phospholipid headgroup in the presence of water and its partial charge distribution. The angle between the headgroup and the acyl chains is nearly perpendicular, suggesting that in the absence of other interactions the inositol ring would lie flat along the cytoplasmic surface of the plasma membrane. Next, we employed hybrid quantum mechanics/molecular mechanics (QM/MM) simulations to investigate the protonation state of PIP2 and its interactions with magnesium or calcium. We test the hypothesis suggested by prior experiments that binding of magnesium to PIP2 is mediated by a water molecule that is absent when calcium binds. These results may explain the selective ability of calcium to induce the formation of PIP2 clusters and phase separation from other lipids.

Magnesium-Induced Nucleophile Activation in the Guanylyltransferase mRNA Capping Enzyme

The mRNA guanylyltransferase, or mRNA capping enzyme, cotranscriptionally caps the 5'-end of nascent mRNA with GMP during the second reaction in a set of three enzymatic reactions that result in the formation of an N7-methylguanosine cap during mRNA maturation. The mRNA capping enzyme is characterized, in part, by a conserved lysine nucleophile that attacks the α-phosphorus atom of GTP, forming a lysine-GMP intermediate. Experiments have firmly established that magnesium is required for efficient intermediate formation but have provided little insight into the requirement's molecular origins. Using empirical and thermodynamic integration pK(a) estimates, along with conventional molecular dynamics simulations, we show that magnesium binding likely activates the lysine nucleophile by increasing its acidity and by biasing the deprotonated nucleophile into conformations conducive to intermediate formation. These results provide additional functional understanding of an important enzyme in the mRNA transcript life cycle and allow functional analogies to be drawn that affect our understanding of the metal dependence of related superfamily members.

The vWFA2 domain of type VII collagen is responsible for collagen binding

Type VII collagen (Col7) is the major component of anchoring fibrils and very important for skin integrity. This is emphasized by the Col7 related skin blistering diseases dystrophic epidermolysis bullosa and epidermolysis bullosa acquisita. Structural data that provides insights into the interaction network of Col7 and thus providing a basis for a better understanding of the pathogenesis of the diseases is missing.We proved that the von-Willebrand-factor A like domain 2 (vWFA2) of Col7 is responsible for type I collagen binding. The interaction has a KD value of 90μM as determined by SPR and is enthalpy driven as derived from the van’t Hoff equation. Furthermore, a hitherto unknown interaction of this domain with type IV collagen was identified. The interaction of vWFA2 with type I collagen is sensitive to the presence of magnesium ions, however, vWFA2 does not contain a magnesium binding site thus magnesium must bind to type I collagen.A lysine residue has been identified to be crucial for type I collagen binding. This allowed localization of the binding site. Mutational analysis suggests different interaction mechanisms in different species and that these interactions might be of covalent nature.

Zinc inhibits magnesium-dependent migration of human breast cancer MDA-MB-231 cells on fibronectin

Metastasis is the major cause of breast cancer mortality. The strength of cell adhesion to extracellular matrix is critical to cancer cell migration. Integrins, the primary mediators of cell to extra-cellular matrix adhesion, contain distinct divalent cation-binding sites. Binding of manganese and magnesium is vital to integrin-mediated cancer cell adhesion and migration. We hypothesized that zinc, a divalent cation, can modulate breast cancer metastasis through interfering with these divalent cation-dependent integrin-mediated cancer cell adhesion and migration. MDA-MB-231 cells were cultured in a zinc-depleted medium supplemented with 0 (control), 2.5, 5, 10, 25 and 50 μM of zinc to mimic severe zinc-deficiency, moderate zinc-deficiency, adequate zinc and three levels of zinc-supplementation: low-, moderate- and high-levels of zinc-supplementation, respectively. Zinc treatments had no effect on cellular zinc concentration, cell number and cell viability. Zinc at 5–50 μM reduced migration distance of MDA-MB-231 cells on fibronectin by 43–86% and migration rate on fibronectin by 72–90%. Zinc induced a dose-dependent inhibition of cell adhesion to fibronectin (R2=−0.98). Zinc at 10–50 μM reduced magnesium-facilitated cell adhesion to fibronectin in a dose-dependent manner (R2=−0.90). However, zinc had no effect on manganese-facilitated cell adhesion to fibronectin. Zinc at 5–50 μM caused rounding of the normally elongated, irregular-shaped MDA-MB-231 cells and disappearance of F-actin. Anti-integrin α5- and β1-subunit blocking antibodies inhibited magnesium-facilitated cell adhesion to fibronectin by 95 and 99%, respectively. In summary, zinc inhibited MDA-MB-231 cell migration on fibronectin by interfering with magnesium-dependent integrin-, likely integrin α5/β1-, mediated adhesion.

The human "magnesome": detecting magnesium binding sites on human proteins

BackgroundMagnesium research is increasing in molecular medicine due to the relevance of this ion in several important biological processes and associated molecular pathogeneses. It is still difficult to predict from the protein covalent structure whether a human chain is or not involved in magnesium binding. This is mainly due to little information on the structural characteristics of magnesium binding sites in proteins and protein complexes. Magnesium binding features, differently from those of other divalent cations such as calcium and zinc, are elusive. Here we address a question that is relevant in protein annotation: how many human proteins can bind Mg2+? Our analysis is performed taking advantage of the recently implemented Bologna Annotation Resource (BAR-PLUS), a non hierarchical clustering method that relies on the pair wise sequence comparison of about 14 millions proteins from over 300.000 species and their grouping into clusters where annotation can safely be inherited after statistical validation.ResultsAfter cluster assignment of the latest version of the human proteome, the total number of human proteins for which we can assign putative Mg binding sites is 3,751. Among these proteins, 2,688 inherit annotation directly from human templates and 1,063 inherit annotation from templates of other organisms. Protein structures are highly conserved inside a given cluster. Transfer of structural properties is possible after alignment of a given sequence with the protein structures that characterise a given cluster as obtained with a Hidden Markov Model (HMM) based procedure. Interestingly a set of 370 human sequences inherit Mg2+ binding sites from templates sharing less than 30% sequence identity with the template.ConclusionWe describe and deliver the "human magnesome", a set of proteins of the human proteome that inherit putative binding of magnesium ions. With our BAR-hMG, 251 clusters including 1,341 magnesium binding protein structures corresponding to 387 sequences are sufficient to annotate some 13,689 residues in 3,751 human sequences as "magnesium binding". Protein structures act therefore as three dimensional seeds for structural and functional annotation of human sequences. The data base collects specifically all the human proteins that can be annotated according to our procedure as "magnesium binding", the corresponding structures and BAR+ clusters from where they derive the annotation (http://bar.biocomp.unibo.it/mg).

The role of salt concentration and magnesium binding in HIV-1 subtype-A and subtype-B kissing loop monomer structures

The subtype-B monomers of the human immunodeficiency virus type-1 (HIV-1) have experimentally been shown to dimerize at high salt concentration or in the presence of magnesium, while the dimerization of the subtype-A monomers requires magnesium binding at the G273 or G274 phosphate groups regardless of salt concentration. We used explicit solvent molecular dynamics (MD) simulations to investigate the conformational changes in subtype-A and -B monomers in different salt concentrations, and we found that our MD simulation results are consistent with those of experiments. At low salt concentration, hairpin loop structures of both subtypes were deformed and bases in the hairpin loop were turned inside. At high salt concentrations, the subtype-B monomer maintained the hairpin loop shape and most bases in the hairpin loop pointed out, while the subtype-A monomer showed a severe deformation. We also found that the flanking bases in the subtype-B stabilize the hairpin loop, while the flanking base G273 in the subtype-A caused a significant deformation. However, a bound magnesium ion at the G273 or G274 phosphate groups controlled the behavior of the G273 base and prevented the subtype-A monomer from deformation. We also applied restraints to both subtypes to examine the role of high salt concentration or magnesium binding. While restraints were applied, both subtypes at 0 M salt concentration maintained their shapes. However, when restraints were removed, they deformed significantly. Therefore, we suggest that the dimerization of both subtypes requires the proper conformation of the monomers which is induced by the appropriate salt strength and magnesium ion binding.

Cell wall structure of selected yeast species as a factor of magnesium binding ability

This study was undertaken to determine the magnesium ion biosorption ability of the C. utilis and S. cerevisiae yeast species during cultivation in model media supplemented with magnesium. The mannoprotein and β-glucan content in the investigated yeast cell wall were analyzed because of the essential function of yeast cell wall structural components in metal ion binding. At the same time, an observation of yeast cells with the use of a transmission electron microscopy (TEM) was performed. The S. cerevisiae No. 1 yeast demonstrated the largest magnesium cation biosorption capacity. The magnesium content in biomass of S. cerevisiae No. 1 was about 16 mg Mg2+/g of dry substance after living cell incubation in MgSO4 solution and about 18 mg Mg2+/g of dry substance after pasteurized biomass incubation in YPD medium supplemented with magnesium ions. The tested yeast strains differed in mannoprotein and β-glucan content in the cell wall. The cell wall of S. cerevisiae 102, coming from YPD + Mg2+ medium, contained the greatest amount of glycoproteins (approx. 66 % adjusted to a total sugar basis). The cell wall of C. utilis ATTC 9950 yeast incubated under the same conditions was composed mainly of β-glucans (approx. 78 %) with prevailing β-(1,6)-glucans in this glucose polymer fraction (approx. 53 %). In S. cerevisiae No. 1 and C. utilis yeasts, higher degrees of magnesium ion binding were observed in the presence of higher β-glucan content in the cell wall structure, whereas in S. cerevisiae, 102 cells the magnesium ion adsorption was determined mainly on the grounds of mannoprotein presence. The process of yeast cell pasteurization increased the magnesium ion binding ability in the tested fungi strains as a result of cell wall structure loosening.

Importance of Length and Sequence Order on Magnesium Binding to Surface-Bound Oligonucleotides Studied by Second Harmonic Generation and Atomic Force Microscopy

The binding of magnesium ions to surface-bound single-stranded oligonucleotides was studied under aqueous conditions using second harmonic generation (SHG) and atomic force microscopy (AFM). The effect of strand length on the number of Mg(II) ions bound and their free binding energy was examined for 5-, 10-, 15-, and 20-mers of adenine and guanine at pH 7, 298 K, and 10 mM NaCl. The binding free energies for adenine and guanine sequences were calculated to be -32.1(4) and -35.6(2) kJ/mol, respectively, and invariant with strand length. Furthermore, the ion density for adenine oligonucleotides did not change as strand length increased, with an average value of 2(1) ions/strand. In sharp contrast, guanine oligonucleotides displayed a linear relationship between strand length and ion density, suggesting that cooperativity is important. This data gives predictive capabilities for mixed strands of various lengths, which we exploit for 20-mers of adenines and guanines. In addition, the role sequence order plays in strands of hetero-oligonucleotides was examined for 5'-A(10)G(10)-3', 5'-(AG)(10)-3', and 5'-G(10)A(10)-3' (here the -3' end is chemically modified to bind to the surface). Although the free energy of binding is the same for these three strands (averaged to be -33.3(4) kJ/mol), the total ion density increases when several guanine residues are close to the 3' end (and thus close to the solid support substrate). To further understand these results, we analyzed the height profiles of the functionalized surfaces with tapping-mode atomic force microscopy (AFM). When comparing the average surface height profiles of the oligonucleotide surfaces pre- and post- Mg(II) binding, a positive correlation was found between ion density and the subsequent height decrease following Mg(II) binding, which we attribute to reductions in Coulomb repulsion and strand collapse once a critical number of Mg(II) ions are bound to the strand.

RAG-1 (Recombination Activating Gene-1) protein is closely related to herpes virus recombinases: Implications for the origins of the acquired immune system. (105.20)

Abstract The acquired immune system results from recombination of germ-line DNA V(D)J signal sequences by the RAG-1 recombinase. RAG-1 is a dde magnesium binding recombinase related to both retroviral integrases and transposases. However the closest phylogenetic relative to RAG-1 appears to be a family of recombinases encoded by the herpesviridae termed the DBP (DNA binding proteins) that catalyze strand exchange and possibly other recombination events during herpes virus replication. Both the dde magnesium binding triad and the nonamer binding regions of RAG-1 are present and conserved in the DBP. These observations suggest that initially RAG-1 arose from an ancient insertion of a herpes-like infectious agent into a deuterostome genome prior to the origins of the acquired immune system. The author proposes a model in which the primordial RAG-1 recombinase was selected through its ability to promote viral immune responses through innate immune receptors in ancient deuterostomes prior to the origins of V(D)J recombination.

Cooperative Modulation of Mineral Growth by Prismatic-Associated Asprich Sequences and Mg(II)

Cooperative effects of magnesium ions and acidic polypeptides originating from a family of proteins known as Asprich (mollusk Atrina rigida) were studied. In our previous studies, these two acidic polypeptides were found to be effective in controlling the morphology of the calcium carbonate mineral, the main inorganic constituent of prismatic layer of the mollusk shell. Since these Asprich sequences are believed to contain a putative magnesium binding domain, the morphology-controlling effects were further investigated with the addition of magnesium ions. The mineral morphology was dramatically changed by the combined influence of each polypeptides and the magnesium ions, substantiating the recognized importance of magnesium in the formation of calcium carbonate-based biominerals.

Cation Binding and Screening Effects on Single-Stranded Helix Formation in Poly(A) RNA

The RNA homopolymer poly(A) forms single-stranded helices due to base-stacking, the mechanical properties of which may affect the biological function of naturally-occurring A-repeat sequences. Since RNA is a polyanion, screening and binding by cations enhances this helix formation by shielding charges along its backbone from each other.The poly(A) single helix may be disrupted by application of tension, as by optical tweezers, allowing the energetics of helix formation to be estimated in addition to the mechanical properties of the helical and randomly-coiled states.Using optical tweezers, we measured the force-extension behavior of poly(A) under a variety of buffer conditions, finding that the effect of sodium is consistent with Debye screening but that magnesium and other Group II cations are likely to be binding and forming inner-sphere (direct) contacts with poly(A), stabilizing the helix more than would be expected for screening alone. The mode of binding appears to be dependent on cation species, with distinct differences between binding by strontium and by magnesium and calcium.To obtain quantitative information from these force-extension measurements, we have developed a new model for the force-extension behavior of poly(A) and similar helicogenic polymers, which unlike previous models takes into account Debye screening effects and the semiflexible nature of the helical state. Fitting this model to our data, we obtain estimates of helix formation energies and find evidence that magnesium binding distorts the poly(A) single helix into a mechanically distinct state.

Glass-Like Behavior of Magnesium Ions Inside RNA APTamers

RNA is known to be strongly dependent on Magnesium for stability. In this computational study, we explore the effects of Magnesium concentration on the SAM I riboswitch with several microsecond-plus simulations at varying concentrations. Individual Magnesium binding events are shown to influence the RNA conformation through tertiary bridging interactions. We characterize the behavior of Magnesium near RNA for use in structure-based models, and note that it is glassy.

Binding of Calcium, Magnesium, and Target Peptides to Cdc31, the Centrin of Yeast Saccharomyces cerevisiae

Cdc31, the Saccharomyces cerevisiae centrin, is an EF-hand calcium-binding protein essential for the cell division and mRNA nuclear export. We used biophysical techniques to investigate its calcium, magnesium, and protein target binding properties as well as their conformations in solution. We show here that Cdc31 displays one Ca(2+)/Mg(2+) mixed site in the N-terminal domain and two low-affinity Ca(2+) sites in the C-terminal domain. The affinity of Cdc31 for different natural target peptides (from Kar1, Sfi1, Sac3) that we obtained by isothermal titration calorimetry shows weakly Ca(2+), but also Mg(2+) dependence. The characteristics of target surface binding were shown to be similar; we highlight that the 1-4 hydrophobic amino acid motif, in a stable amphipathic α-helix, is critical for binding. Ca(2+) and Mg(2+) binding increase the α-helix content and stabilize the structure. Analysis of small-angle X-ray scattering experiments revealed that N- and C-terminal domains are not individualized in apo-Cdc31; in contrast, they are separated in the Mg(2+) state, creating a groove in the middle of the molecule that is occupied by the target peptide in the liganded form. Consequently, Mg(2+) seems to have consequences on Cdc31's function and could be important to stimulate interactions in resting cells.

Structure and binding specificity of the receiver domain of sensor histidine kinase CKI1 from Arabidopsis thaliana

Multistep phosphorelay (MSP) signaling mediates responses to a variety of important stimuli in plants. In Arabidopsis MSP, the signal is transferred from sensor histidine kinase (HK) via histidine phosphotransfer proteins (AHP1-AHP5) to nuclear response regulators. In contrast to ancestral two-component signaling in bacteria, protein interactions in plant MSP are supposed to be rather nonspecific. Here, we show that the C-terminal receiver domain of HK CKI1 (CKI1(RD) ) is responsible for the recognition of CKI1 downstream signaling partners, and specifically interacts with AHP2, AHP3 and AHP5 with different affinities. We studied the effects of Mg²⁺, the co-factor necessary for signal transduction via MSP, and phosphorylation-mimicking BeF₃⁻ on CKI1(RD) in solution, and determined the crystal structure of free CKI1(RD) and CKI1(RD) in a complex with Mg²⁺. We found that the structure of CKI1(RD) shares similarities with the only known structure of plant HK, ETR1(RD) , with the main differences being in loop L3. Magnesium binding induces the rearrangement of some residues around the active site of CKI1(RD) , as was determined by both X-ray crystallography and NMR spectroscopy. Collectively, these results provide initial insights into the nature of molecular mechanisms determining the specificity of MSP signaling and MSP catalysis in plants.

Mutational analysis of the ribC gene of Bacillus subtilis

The nucleotide sequence of the ribC gene encoding the synthesis ofbifunctional flavokinase/flavine adenine nucleotide (FAD) synthetase in Bacillus subtilis have been determined in a family of riboflavin-constitutive mutants. Two mutations have been found in the proximal region of the gene, which controls the transferase (FAD synthase) activity. Three point mutations and one double mutation have been found (in addition to the two mutations that were detected earlier) in the distal region of the gene, which controls the flavokinase (flavin mononucleotide (FMN) synthase) activity. On the basis of all data known to date, it has been concluded that the identified mutations affect riboflavin and ATP binding sites. No mutations have been found in the PTAN conserved sequence, which forms the magnesium and ATP common binding site and is identical for organisms of all organizational levels, from bacteria too humans.

Briefly Bound to Activate: Transient Binding of a Second Catalytic Magnesium Activates the Structure and Dynamics of CDK2 Kinase for Catalysis

We have determined high-resolution crystal structures of a CDK2/Cyclin A transition state complex bound to ADP, substrate peptide, and MgF(3)(-). Compared to previous structures of active CDK2, the catalytic subunit of the kinase adopts a more closed conformation around the active site and now allows observation of a second Mg(2+) ion in the active site. Coupled with a strong [Mg(2+)] effect on in vitro kinase activity, the structures suggest that the transient binding of the second Mg(2+) ion is necessary to achieve maximum rate enhancement of the chemical reaction, and Mg(2+) concentration could represent an important regulator of CDK2 activity in vivo. Molecular dynamics simulations illustrate how the simultaneous binding of substrate peptide, ATP, and two Mg(2+) ions is able to induce a more rigid and closed organization of the active site that functions to orient the phosphates, stabilize the buildup of negative charge, and shield the subsequently activated γ-phosphate from solvent.