How native and non-native cations bind and modulate the properties

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How native and non-native cations bind and modulate the properties of GTP/ATP Todor Dudev, Cedric Grauffel, Shang-Te Danny Hsu, and Carmay Lim J. Chem. Theory Comput., Just Accepted Manuscript • DOI: 10.1021/acs.jctc.8b00259 • Publication Date (Web): 16 May 2018 Downloaded from http://pubs.acs.org on May 19, 2018

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Journal of Chemical Theory and Computation

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How native and non-native cations bind and modulate the properties of GTP/ATP Todor Dudev,1,* Cédric Grauffel,2 Shang-Te Danny Hsu,3 and Carmay Lim2,4,* 1

Faculty of Chemistry and Pharmacy, Sofia University, Sofia 1164, Bulgaria

2

Institute of Biomedical Sciences, Academia Sinica, Taipei 11529, Taiwan

3

Institute of Biological Chemistry, Academia Sinica, Taipei 11529, Taiwan

4

Department of Chemistry, National Tsing Hua University, Hsinchu 300, Taiwan

ABSTRACT Adenosine triphosphate (ATP) and guanosine triphosphate (GTP) exist in physiological solution mostly bound to cations. Interestingly, their cellular Mg2+-bound forms have been shown to bind Li+, a first-line drug for bipolar disorder. However, solution structures of NTP/NDP (N = A or G) bound to Li+ and/or Mg2+ have not been solved, thus precluding knowledge of how the native Mg2+-bound cofactor conformation changes upon binding nonnative Li+ and/or switching its environment from aqueous solution to proteins. Using wellcalibrated methods that reproduce experimental structural and thermodynamic parameters of several Mg2+/Li+-nucleotide complexes, we show that the native NTP/NDP-Mg2+ cofactor adopts a “folded” conformation in water that remains unperturbed upon Li+ binding. We further show that the ATP-binding pockets of receptors such as P2X is complementary in shape to the “folded” ATP-Mg2+ solution structure, whereas the elongated GTP-binding pockets found in G-proteins necessitate the GTP-Mg2+ cofactor to undergo a conformational change from its “folded” conformation in solution to an extended one upon G-protein binding. Implications of the findings on how Li+, in its bound state, can manifest its therapeutic effects are discussed.

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Evolution has bestowed biological functions to about two dozen metal ions based on their physicochemical properties and bioavailability.1 The most common biogenic metal ions are Na+, K+, Mg2+, Ca2+, Zn2+, and redox-active transition metal cations that can exist in multiple oxidation states such as Mn2+/3+/4+,2 Fe2+/3+/4+,3 Co2+/3+,4 Ni+/2+/3+,5 Cu+/2+6 and Mo3+/4+/5+.7 Non-biogenic cations such as Hg2+, Pb2+, Al3+, and Tl+/Tl3+ can enter an organism and disrupt cellular functions, causing pathological conditions,8 whereas Li+, Sr2+, and Ga3+ may exert beneficial effects on the host organism. Notably, Li+, not known to have essential biological functions in mammals, is used to treat mental illnesses especially bipolar disorder.9, 10 It is being considered as a therapeutic agent for damage caused by traumatic brain injury11 and for treating chronic neurodegenerative diseases such as Alzheimer’s, Parkinson’s, and Huntington’s diseases.9,

12

Despite the plethora of information on lithium’s clinical and

pharmacodynamical effects, its precise mechanism of therapeutic action remains elusive, warranting further study. Different modes of lithium’s action have been hypothesized,10,13 which altogether underscore the complex pharmacodynamic actions of Li+ through its ability to modulate diverse signaling pathways. Free hydrated Li+ has been postulated to compete with Na+ and Mg2+. Bipolar disorder patients have abnormally elevated intracellular Na+ concentration.14, 15 By entering the cytosol via Na+ channels and Na+/Ca2+ exchanger, Li+ accumulates in the cytosol, leading to a reduction of Na+ and Ca2+ intracellular concentration, which in turn reduces the cell excitability and normalizes the neuronal activity in bipolar disorder patients.15 Free hydrated Li+ has also been postulated to displace Mg2+ in certain enzymes that are overexpressed in mentally ill patients such as glycogen synthase kinase 3β and inositol mono/polyphosphatase.10,

13, 16-19

Inhibition of these signal-transducing enzymes

eventually normalizes the cell signaling in bipolar disorder patients. Instead of competing with Mg2+, Li+ can exert its therapeutic effect by co-binding with Mg2+ to adenosine triphosphate (ATP) to form a bimetallic ATP-Mg-Li complex that can activate purine receptors (P2X/P2Y) like the native cofactor:20, 21 When bound to either ATPMg or ATP-Mg-Li, ligand-gated ion channels, P2X, open to allow extracellular Ca2+ inside the cell, whereas G-protein coupled receptors, P2Y, trigger Ca2+ release from intracellular stores.21 Subsequent calculations showed that Li+ binding did not alter the native [Mg-ATP]2– solution conformation, hence the bimetallic complex could fit in the host protein’s ATPbinding site.22


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3 Guanosine di/triphosphate (GDP/GTP) are also involved in cell signaling. They regulate the activity of heterotrimeric guanine nucleotide-binding proteins (G-proteins), molecular switches orchestrating signal transduction from the extracellular matrix to the respective intracellular effectors.23, 24 Central to the G-protein activity is the interplay between the GDP and GTP binding: In the resting, inactive state, the G-protein’s α subunit harbors GDP. It becomes activated upon binding Mg2+_bound GTP, whose hydrolysis to GDP deactivates the G-protein and returns it to the resting state. G-proteins are hyperactive/overexpressed in bipolar disorder patients, but administering lithium salts suppressed their activities and normalized cell signaling.10,

17, 25-27

Lithium has been postulated to manifest its therapeutic

action by competing and replacing Mg2+, thus inhibiting the G-protein,10, 17 but calculations indicate that this is unlikely if Mg2+ were phosphate-bound.19 How Li+ binding to GTP-Mg2+ would affect the G-protein–metal-bound-GTP recognition has not been considered. Notably, bimetallic NTP/NDP-Mg-Li (N = A/G) structures in water/proteins have not been solved. This raises the following intriguing, fundamental questions: 1. How does the nucleotide base and the loss of the terminal phosphate upon NTP hydrolysis affect the metal-binding mode? 2. Are the NTP-binding pockets of proteins that are targeted by Li+ complementary in shape to the most stable solution conformation of the nucleotide-metal complexes? 3. Do protein ligands interact with the bimetallic NTP-Mg-Li complexes as well as the native Mg2+-bound cofactor? 4. How does Li+ binding to NTP-Mg modulate the nucleotide’s hydrolytic stability? To address these questions, we modeled Li+ and/or Mg2+-bound NTP/NDP complexes with varying (i) metal composition (mono/binuclear), (ii) metal-binding moieties (phosphate/base) and metal-binding modes where one (monodentate), two (bidentate) or three (tridentate) phosphates coordinate the cation, and (iii) solvent exposure. The preferred metalbinding modes of NTP/NDP and the thermodynamic parameters for various reactions involving the nucleotide-metal complexes were evaluated using density functional theory and polarizable continuum model calculations (see Methods). By comparing the nucleotide conformations in the solution structures of mono/bimetallic complexes to those in the crystal structures of Li+ target proteins, we reveal how Li+ co-binding with Mg2+ affects the nucleotide structure and properties, which impact the signal transduction mechanisms of related biological systems.


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METHODS Modeling Metal-Nucleotide Complexes. At ambient pH in water, NDP and NTP exist predominantly as fully deprotonated anions; i.e., NDP3– and NTP4–, as the pKa values of the constituent di/triphosphate moieties are below 7 and decrease further upon cation coordination.28-30 In aqueous solution, ATP and ADP form 1:1 complexes31, 32 with Mg2+ and the phosphate groups coordinate the cation directly,33 hence, 1:1 inner-sphere nucleotidecation complexes were modeled. Since Mg2+ is usually hexacoordinated in proteins and in complexes with organic ligands,34, 35 it was assumed to be hexacoordinated in the NTP/NDP complexes. The smaller Li+ prefers to be tetracoordinated in its complexes,35, 36 hence it was tetracoordinated to the Mg2+-bound cofactors. All the complexes contain five water molecules, irrespective of the metal-binding mode. Thus, in the mononuclear complexes, five water molecules were retained in the first shell when Mg2+ was monodentately bound to the nucleotide, but one/two of these water molecules were transferred to the second shell when Mg2+ was bi/tridentately bound. The Lys side chain and peptide backbone group, which are commonly seen interacting with the metal-bound nucleotides in protein structures, were modeled as n-pentylammonium

(CH3CH2CH2CH2CH2NH3+)

and

N-methylacetamide

respectively. All constructs were built using GaussView.

(CH3CONHCH3),

37

Geometry Optimization. The M062X/6-311++G(d,p) method, which was found to be the most efficient in reproducing the experimentally determined structural parameters of several Mg2+ and Li+ complexes in previous work,22 was used to optimize the geometry of each metal complex in water (ε = 78) using Gaussian 09e.38 For each metal-binding mode (mono, bi, or tridentate) or site (di/triphosphate, adenine/guanine), we modeled several structures, trying to maximize the number of favorable intramolecular interactions − the optimized structure with the lowest energy was chosen for further analyses. To mimic protein binding sites of varying solvent exposure, geometry optimizations were carried out using lower effective ε values of 10−30 using Gaussian’s default probe radius of water. For each of the lowest-energy complexes, vibrational frequencies were evaluated at the same M062X/6311++G(d,p) level – no imaginary frequency was found in any of the optimized structures. Metal-Binding Mode Determination. To determine the most preferred binding mode of a given cation to NTP in water, we computed the probability of each metal-binding


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5 mode according to the Boltzmann distribution. The solution electronic energies, Eel, in the Boltzmann formula were corrected by single-point energy calculations using 16 functionals (B-LYP, B3-LYP, B3-LYP+GD3, PBE1-PBE, WB97XD, M05, M05-2X, M06-L, M06, M06-2X, M11-L, M11, MN12-L, MN12-SX, N12, and N12-SX), three basis sets (6311++G(d,p), 6-311++G(2d,2p), 6-311++G(3d,p)) and the SMD solvation model.39 Free Energy Calculations. The free energy of the metal-bound NTP/NDP complex relative to the lowest free energy structure was computed according to ∆∆G = ∆∆Eel + ∆∆Eth − T∆∆S

(1)

where the thermal energies (Eth) and entropies (S) were computed from standard statistical mechanical formulas40 using frequencies scaled by an empirical factor of 0.979.41 To determine an optimal method for the single-point Eel calculations, we used various methods (see above) to compute Eel and the Boltzmann-weighted solution free energies for [ATP-Mg] + [GTP-Mg-Li] → [GTP-Mg] + [ATP-Mg-Li]

(2)

[ADP-Mg] + [GDP-Mg-Li] → [GDP-Mg] + [ADP-Mg-Li]

(3)

This is because Li+ equilibrium dissociation constants for NTP/NDP-Mg21 at 283K have been measured yielding experimental free energies for eq 2 (−0.62±0.14 kcal/mol) and eq 3 (−0.42±0.20 kcal/mol). Among the 48 methods tested, the PBE1PBE functional combined with the 6-311++G(2d,2p) method best reproduced the experimental free energies with computed values of −0.58 and −0.84 kcal/mol, respectively (Supplementary Tables S1 and S2). Thus, it was used to compute the relative free energies of the various metal-nucleotide complexes and those for the hydrolytic/ligand-binding reactions studied. NMR experiments. All NMR samples contain 1 mM of NTP/NDP buffered in 20 mM sodium phosphate (pH 7.4) with 10 % D2O (v/v) for deuterium signal locking. 1mM of the NTP/NDP was used because at higher concentrations, the nucleotide may undergo selfassociation via aromatic-ring stacking of the bases since the presence of cations reduce the repulsive effect of the negatively charged triphosphate.42 For each NTP/NDP, four combinations were made: (i) NTP/NDP alone, and the nucleotide with (ii) 1 mM LiCl, (iii) 1 mM MgCl2, or (iv) 1 mM LiCl and 1 mM MgCl2. The 1H, 7Li, and 31P spectra of individual samples were collected at 298 K using a 500 MHz NMR spectrometer (AVANCE III, Bruker BioSpin, Germany) equipped with a cryo-cooled Prodigy broadband probe head. The NMR data were processed and analyzed using Topspin 3.2 (Bruker BioSpin, Germany).


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RESULTS Metal-Binding Modes of NTP in Water. To determine how the nucleotide base affects the metal-binding mode, we optimized different constructs with varying metal-binding modes/sites and computed their relative solution free energies, ∆∆G. The Mg2+-binding free energy to the guanine base is much less favorable than that to the triphosphate moiety (Supplementary Figure S1), indicating that the cation interacts exclusively with the polyphosphate moiety. The ∆∆G in Supplementary Figure S1 and Boltzmann populations of each metal-binding mode (Supplementary Figure S2) show that the tridentate [NTPMg(αβγ)]2– structures with Mg2+ bound by all three phosphate groups are most stable, as found in QM/MM simulations of [GTP-Mg]2–,43 whereas Li+ is predominantly bound by the βγ phosphates and is linked to Mg2+ by a hydroxide in the bimetallic [NTP-Mg(αβγ)OHbridge-Li(βγ)]2– complexes (Figure 1).

Figure 1. Fully optimized M062X/6-311++G(d,p) structures of the most populated [NTPMg]2– and [NTP-Mg-OH-Li]2– complexes in water, oriented to best depict the metal-binding modes; the base in the bimetallic complexes is hidden behind the ribose. Metal-Binding Modes of NDP in Water. With one less phosphate in NDP compared to NTP, what are the preferred metal-binding mode(s) and can Li+ still form a hydroxide-bridged

[NDP-Mg-OH-Li]–

complex?

To

address

this,

we

optimized

mono/binuclear structures with different metal-binding modes/sites (Supplementary Figure ACS Paragon Plus Environment

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7 S3) and computed their Boltzmann populations to determine the predominant coordination mode for each type of NDP-metal complex (Supplementary Figure S4). The resulting structures show that Mg2+ still prefers to bind to the NDP diphosphate rather than the base, and Li+ can co-bind with Mg2+ via a hydroxide bridge to NDP. Both cations are predominantly bound by the αβ phosphates in the mono/binuclear complexes (Figure 2).

Figure 2. Fully optimized M062X/6-311++G(d,p) structures of the most stable [NDP-Mg]– and [NDP-Mg-OH-Li]– complexes in water, oriented to depict the metal-binding modes clearly; the base in the bimetallic complexes is hidden behind the ribose. Nucleotide-Metal Conformations in Water. Interestingly, the nucleotides adopted a “folded” conformation upon cation binding where the metal-bound polyphosphate moiety was folded towards the ribose ring, regardless of the metal-binding mode/site (Figures 1 and 2). Notably, binding of Li+ to NTP/NDP-Mg2+ did not significantly alter the overall structure of the native cofactor, whose heavy atoms exhibited a mean root-mean-square deviation (RMSD) from those of the bimetallic structures by ≤ 0.6 Å. It also did not alter the net charge of [NTP-Mg]2–/[NDP-Mg]–, as the water molecule bridging the two cations is likely to be deprotonated: The free energy for deprotonating the bridging water molecule in the ADPMg(αβ)-H2O-Li(β) complex is –20 kcal/mol, whereas the corresponding numbers for the bimetallic GTP and GDP complexes are –17 and –18 kcal/mol, respectively. Experimental Validation of the Nucleotide-Metal Solution Conformations. Our calculations agree with

17

O NMR,44

31

P NMR,31,

45

and Raman/infrared studies30


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8 showing that the predominant isomer in water is tridentate [NTP-Mg(αβγ)]2– and bidentate [NDP-Mg(αβ)]–. The tridentate Mg2+-binding mode to NTP in water supports a “folded” conformation, as Mg2+ cannot simultaneously bind to all three phosphates if the NTP conformation were extended. Consistent with experiment,21 the Li+ dissociation free energy from GTP/GDP-Mg2+ was computed to be more favorable than that from ATP/ADP-Mg2+ at 283K (see Methods), lending indirect support for the Li+-binding modes found. To assess whether lithium’s therapeutic dosage (0.6−1.0 mM) negligibly perturbs the native NTP/NDP–Mg2+ structure, 1 mM Li+ and physiological (1 mM) concentrations of Mg2+ and NTP/NDP were used to determine the effects of the cation on the nucleotide conformations based on the 7Li/31P chemical shifts. The results in Figure 3 show three wellresolved 31P resonances with narrow lines corresponding to the NTP α, β, and γ phosphates, and two peaks corresponding to the NDP α and β phosphates. Adding 1 mM Mg2+ to NTP/NDP induced severe line-broadening for the β and γ phosphates and marked chemical shift up-field, consistent with previous studies.21 In contrast, adding 1 mM Li+ to free/Mg2+bound NTP/NDP induced negligible linewidth and chemical shift changes (< 0.1 ppm, Supplementary Table S3) to the nucleotide

31

P resonances, suggesting that Li+ negligibly

perturbed the native NTP/NDP–Mg2+ solution structure. Although the NMR experiments cannot provide direct proof of a bridging hydroxide in the bimetallic complexes, they nonetheless suggest its presence. Under our experimental conditions, a significant proportion of the bimetallic complex is expected, as the Li+ equilibrium dissociation constants from Mg2+-NTP/NDP in water are in the mM range.21 Lithium’s positive charge near the Mg2+-bound polyphosphate moiety would be expected to significantly perturb the phosphate chemical environment and thus the

31

P chemical shift.

+

However, the negligible Li -induced chemical shift perturbations of the NTP/NDP–Mg2+ 31P resonances is consistent with neutralization of the lithium’s charge by the bridging hydroxide in the bimetallic complexes.


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Figure 3.

31

P NMR analyses of Li+ and/or Mg2+ binding to NTP/NDP. The molar

concentrations of NTP/NDP, Li+, and Mg2+ were all set to 1 mM. The spectra were collected at 298 K and 11.7 Tesla (corresponding to a Larmor frequency of 202.4 MHz for 31P). Nucleotide Conformational Changes upon Binding to Proteins. Like the native [ATP-Mg]2– cofactor, [ATP-Mg-OH-Li]2– is recognized by purinergic receptors.21 However, [GTP-Mg-OH-Li]2– may bind to G-proteins, as Li+ treatment attenuates G-protein function, which is enhanced in bipolar disorder patients.10 To assess the extent to which the nucleotide conformation in the metal-locked solution structures changes upon binding to Li+ target proteins, we searched the Protein Data Bank (PDB)46 for X-ray structures of ATPreceptors/G-proteins containing NTP. This search yielded eight G-protein structures containing GTP-Mg2+ (PDB codes 1QRA, 2RAP, 4B48, 4KVG, 2XTN, 3WXM, 3FFA and 4K81) and seven P2X structures containing ATP without Mg2+ (PDB codes 4DW1, 5SVL, 5SVK, 5SVM, 5SVP, 5U2H, and 5F1C).


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10 GTP-Mg. All eight [GTP-Mg]2–-bound X-ray structures (Supplementary Figure S5) show Mg2+ bidentately bound to the βγ phosphates of GTP (Figure 4a). In this bidentatebinding mode, GTP adopted an elongated conformation with Pγ–C1 ranging from 9.2–9.8 Å (where C1 is the ribose carbon that is bonded to the base), in contrast to the predominantly “folded” conformation in water where Pγ–C1 is ~6.2 Å. This implies that the “folded” tridentate [GTP-Mg(αβγ)]2– cofactor in solution (Figure 1d), upon entering the G-protein’s nucleotide-binding pocket, has to undergo substantial conformational changes to adopt an elongated bidentate conformation.

Figure 4. (a) GTP conformation in the X-ray structure of p21Ras containing GTP-Mg2+ (PDB entry 1QRA). M062X/6-311++G(d,p) fully optimized structures and relative free energies (in kcal/mol) for ε = 30 of (b) extended and (c) compact [GTP-Mg(βγ)]2– bidentate complexes, and (d) [GTP-Mg(αβγ)]2– tridentate complex. Mg2+ is in magenta. R is the distance between the Pγ and C1 atoms marked by stars. Such a conformational transition from a “folded” tridentate to an extended bidentate structure in the G-protein has several consequences: First, in coming from solution to a partially solvent-shielded protein pocket, [GTP-Mg(αβγ)]2– has to stretch its “folded” tridentate conformation to an extended bidentate one that is higher in free energy. We showed this by optimizing several [GTP-Mg(βγ)]2– bidentate complexes differing in the degree of “extendedness”, as measured by the Pγ–C1 distance, using an effective dielectric constant ε 1 Å were discarded. The Pγ–C1 distances are 10.1 Å and 9.8 Å for Li+ bound by the βγ and γ phosphates, respectively.

Third, trading the rigid tridentate αβγ-structure for a bidentate βγ structure makes the native cofactor more susceptible to further structural changes upon additional cation/ligand interactions: Li+ binding to the tridentate [GTP-Mg(αβγ)]2– did not alter the overall native cofactor’s structure (see above and Figure 6a). However, its binding to the bidentate [GTPMg(βγ)]2– significantly changed the polyphosphate structure (Figure 6b).


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Figure 6. Overlay between native [GTP-Mg]2– (bold) and bimetallic [GTP-Mg-OHbridge-Li]2– (grey) structures optimized at the M062X/6-311++G(d,p) level using ε = 30. (a) Mg2+ (in magenta) tridentately bound to a “folded” nucleotide conformation where the Pγ–C1 distance is 6.15 Å. (b) Mg2+ bidentately bound to an “extended” nucleotide conformation where the Pγ–C1 = 9.92 Å. Water molecules are removed for clarity. ATP-Mg. In contrast to GTP-Mg2+, whose solution conformation changes significantly upon binding G-proteins, the overall ATP conformation in all the P2X crystal structures is similar to that in the computed ATP-Mg2+ solution structures (Figure 7): In the receptorbound structures, ATP adopted a “folded” conformation characterized by a Pγ–C1 distance ranging from 5.3–6.1 Å, as found in the ATP-Mg2+ solution structures. This implies that the shape of the P2X receptor’s ATP-binding pocket is complementary to the predominant ATPMg2+ solution conformation. Since Li+ binding does not alter the [ATP-Mg]2– net charge and conformation (as evidenced by a 0.2 Å RMSD between the mononuclear and binuclear structures and the

31

P NMR chemical shift analysis), the bimetallic [ATP-Mg-OH-Li]2–

complex can also fit in the receptor’s pocket.


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Figure 7. Similarity in the ATP conformations from M062X/6-311++G(d,p) calculations (ε = 30) and from X-ray structures of ATP-bound P2X receptors, three of which are shown. Water molecules are removed for clarity. Mg2+ in magenta, and Li+ in cyan. [ATP-Mg]2– and [ATP-Mg-OH-Li]2– have Similar Affinities for Protein Ligands. The peptide backbone and Asn, Gln, Lys, or Arg side chains are commonly found to interact with the nucleotide polyphosphate moieties in the PDB protein structures (Grauffel & Lim, unpublished results). To assess if these aa ligands lining the binding site interact with native and bimetallic ATP-metal complexes with similar strength, we evaluated the competition between [ATP-Mg]2– and [ATP-Mg-OH-Li]2– for the peptide backbone (modeled by N-methylacetamide) or the Lys side chain (modeled by n-pentyl-ammonium), which are denoted by L by the following reactions: ∆G1bind [ATP-Mg-OH-Li]2–

+ Lq

→

([ATP-Mg-OH-Li]•L)q–2

(4)

([ATP-Mg]•L)q–2

(5)

∆G2bind [ATP-Mg]2–

+ Lq

→

Both native and bimetallic ATP-metal complexes exhibit comparable affinity for the protein ligands assuming they inflict similar conformational changes upon protein binding: their free energies to the peptide backbone group or the Lys side chain differed by < 2 kcal/mol in partially buried or solvent-exposed pockets (Supplementary Figure S6). Hydrolytic Stability of Bimetallic [ATP-Mg-OH-Li]2– vs. Native [ATP-Mg]2–. Although Li+ binding to the native [ATP-Mg]2– cofactor does not seem to obstruct binding to


13


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14 the receptor, could it affect the hydrolytic stability of ATP? To address this, we computed the free energy for [ATP-Mg-OH-Li]2– hydrolysis, [ATP-Mg(αβγ)-OHbridge-Li(βγ)]2– + H2O → [ADP-Mg(αβ)-OHbridge-Li(x)]– + H2PO4– (6) where x denotes αβ, α or β, relative to that for [ATP-Mg]2– hydrolysis, [ATP-Mg(αβγ)]2– + H2O → [ADP-Mg(αβ)]– + H2PO4–

(7)

The free energy differences for different media in Figure 8 show that the coordination modes allowed for Li+ bound to [ADP-Mg]– in the protein pocket could modulate the native cofactor’s hydrolytic stability: If the protein matrix prevented the ADP phosphates from binding Li+ in its preferred solution mode, then Li+ binding to [ATP-Mg]2– reduced the native cofactor’s susceptibility to hydrolysis (∆∆Ghyd > 6 kcal/mol); otherwise, Li+ binding did not significantly affect the hydrolytic stability of [ATP-Mg]2– (|∆∆Ghyd| ~2 kcal/mol).

Figure 8. Free energy difference between equations 6 and 7, which yields ∆∆G (in kcal/mol) for [ATP-Mg(αβγ)-OH-Li(βγ)]2– + [ADP-Mg(αβ)]– → [ADP-Mg(αβ)-OH-Li(x)] – + [ATPMg(αβγ)]2– or different coordination modes of Li+ bound to [ADP-Mg]– as a function of the effective dielectric constant ε (ranging from 10–30). In the [ADP-Mg(αβ)-OH-Li(x)]

–

complexes, the most stable Li+ coordination mode is αβ (bottom left, as in Figure 2b), which


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15 is denoted by green squares. The protein matrix could constrain Li+ to be bound by a less favorable αβ-mode (bottom right, blue squares) or α-phosphate (filled orange circles) or βphosphate (half-filled red circles).


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DISCUSSION ATP and GTP serve as an energy source and play crucial biological roles in signal transduction pathways. In the cell, they are mostly bound by cations, in particular divalent Mg2+ in a 1:1 ratio. Despite their importance, there are currently no atomic NTP/NDP-Mg2+ and NTP/NDP-Mg2+-Li+ solution structures partly because of the poor hydrogen density that hinders nuclear Overhauser effect-based structure determination in water and partly due to the spontaneous hydrolysis of NTP to NDP during crystallization.47 Knowing which of the different metal-bound conformations is lowest in free energy and thus most populated in water is important for studying NTP-Mg2+ hydrolysis in water and NTP/NDP-Mg2+ binding to various protein nucleotide-binding pockets from solution. Using

well-calibrated

methods

that

reproduce

experimental

structural

and

thermodynamic parameters of several Mg2+/Li+-nucleotide complexes, we have obtained solution structures and relative stabilities of Mg2+ and/or Li+ bound to NTP/NDP in various metal-coordination modes/moieties. All 48 methods tested predict predominantly tridentate [NTP-Mg(αβγ)]2– and bidentate [NDP-Mg(αβ)]2– forms in water, consistent with spectroscopic data.30, 31, 44, 45 In contrast, previous studies on ATP hydrolysis in water had assumed high-energy tridentate βγγ48, bidentate βγ49, 50 or αβ47 [ATP-Mg]2– conformations. Notably, the NMR spectra in Figure 3 support Mg2+ coordinated to all three NTP phosphates simultaneously rather than to the αβ and βγ phosphates at different times: If the αβ and βγ modes were populated at different times with a slow interconversion rate, two distinct βphosphate resonances would be expected, but if their interconversion rate were fast, a stronger resonance for the β-phosphate corresponding to the sum of both modes would be expected. Neither two distinct β-phosphate resonances nor a stronger resonance for the βphosphate was observed experimentally, implying that Mg2+ is tridentately bound. All the most stable Mg2+-nucleotide solution structures show a “folded” conformation − an “extended” conformation would be incompatible with the experimentally observed tridentate Mg2+-coordination mode of ATP44 and GTP.30 A corollary of this finding is that Li+ binding to the tridentate [NTP-Mg(αβγ)]2– complex does not alter the native cofactor’s overall charge or conformation. Another corollary is that the predominant metal-nucleotide solution structures enable us to determine the extent of conformational changes that occur upon binding to Li+ target proteins, which has important implications for how Li+, in its bound state, can manifest its therapeutic effects.


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17 One of the targets of Li+ therapy is G-proteins, which are hyperactive/overexpressed in bipolar disorder patients.10, 17, 25-27 Our results suggest that Li+ may affect G-protein function by changing the native cofactor’s polyphosphate conformation in the G-protein. Upon entering the elongated pocket of G-proteins, the tridentate [GTP-Mg(αβγ)]2– solution structure has to undergo significant reorganization to an extended conformation that can coordinate Mg2+ via only two phosphates. This tridentate to bidentate structural change affects bimetallic GTP-Mg-Li and native [GTP-Mg]2– differently: First, changing the bimetallic complex structure has a higher free energy cost than changing the native cofactor structure. Second, in an extended conformation that coordinate Mg2+ bidentately, the polyphosphate conformation of bimetallic GTP-Mg-Li differs from that of native [GTPMg]2–. As a result, G-proteins interact with the bimetallic complex less well than the native [GTP-Mg]2– cofactor, thus lowering the cytosolic levels of activated G-proteins. In contrast to G-proteins, the P2X receptor’s ATP-binding pocket is complementary in shape to the predominant ATP-Mg2+ solution structure. Since Li+ binding neither alters the overall charge nor conformation of the native cofactor, the [ATP-Mg-OH-Li]2– complex can fit geometrically into the protein’s pocket. It can also interact with the protein ligands lining the ATP-binding cavity with virtually the same strength as the native cofactor. Thus, both structural and thermodynamic factors enable the [ATP-Mg-OH-Li]2– complex to be recognized by the host receptor nearly as well as the native cofactor. Furthermore, depending on lithium’s binding mode by ADP in the protein, the hydrolytic stability of [ATP-Mg-OHLi]2– may be comparable to the native cofactor or enhanced, allowing ATP to reside longer in the binding site and elicit protracted receptor response. Indeed, compared to ATP-Mg2+, the bimetallic ATP-Mg-Li complex can activate P2Y receptors comparably, but it prolonged P2X receptor activation.21

CONCLUSIONS Although the ATP and GTP have many common features, Li+ binding to their Mg-bound structures could have opposite effects on their biological mode of action. Due to the different metal-bound ATP and GTP conformations that are recognized by the host protein (“folded” and stretched conformation, respectively), the rigid [ATP-Mg-OH-Li]2– complex is well recognized by P2X/P2Y and can activate both receptors when bound, whereas the [GTP-MgOH-Li]2– complex has to undergo structural deformation that could compromise G-protein activation and function. Nevertheless, in both cases, lithium exerts curative effects on the


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18 host organism, as stimulating the P2X receptors, on one hand, and inhibiting the overexpressed G-proteins, on the other, could help to normalize cell signaling in the central nervous system.

Supporting Information Available: Figure S1 showing the structures and relative energies of the optimized [NTP-Mg]2– and [NTP-Mg-OH-Li]2–. Figure S2 showing the Boltzmann populations of metal-coordination modes in [NTP-Mg]2–. Figure S3 showing the structures and relative energies of the optimized [NDP-Mg]2– and [NDP-Mg-OH-Li]2–. Figure S4 showing the Boltzmann populations of metal-coordination modes in [NDP-Mg]2–. Figure S5 showing conformation of [NTP-Mg]2– in X-ray structures of the PDB. Figure S6 showing that [ATP-Mg]2– and [ATPMg-OH-Li]2– have the same affinity for lysine and backbone ligands. Tables S1 and S2 providing the details of the choice of the protocol for computing energies. Table S3 showing the chemical shifts of NTP/NDP in the presence of metal ions.


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21 39. Marenich, A. V.; Cramer, C. J.; Truhlar, D. G., Universal solvation model based on solute electron density and a continuum model of the solvent defined by the bulk dielectric constant and atomic surface tensions. J. Phys. Chem. B 2009, 113, 6378-6396. 40. McQuarrie, D. A., Statistical Mechanics. Harper and Row: New York, 1976. 41. Alecu, I. M.; Zheng, J.; Zhao, Y.; Truhlar, D. G., Computational Thermochemistry: Scale Factor Databases and Scale Factors for Vibrational Frequencies Obtained from Electronic Model Chemistries. J. Chem. Theory Comput. 2010, 6, 2872-2887. 42. Freisinger, E.; Sigel, R. K. O., From nucleotides to ribozymes—A comparison of their metal ion binding properties. Coord. Chem. Rev. 2007, 251, 1834–1851. 43. Rudack, T.; Xia, F.; Schlitter, J.; Kotting, C.; Gerwert, K., The role of magnesium for geometry and charge in GTP hydrolysis revealed by quantum mechanics/molecular mechanics simulations. Biophys. J. 2012, 103, 293–302. 44. Huang, S. L.; Tsai, M., D., Does the magnesium(II) ion interact with the alphaphosphate of adenosine triphosphate? An investigation by oxygen-17 nuclear magnetic resonance. Biochemistry 1982, 21, 951-959. 45. Prigodich, R. V.; Haake, P., Association Phenomena. 5. Association of Cations with Nucleoside Di- and Triphosphates Studied by 31PNMR. Inorg. Chem. 1985, 24, 89-93. 46. Berman, H. M.; Battistuz, T.; Bhat, T. N.; Bluhm, W. F.; Bourne, P. E.; Burkhardt, K.; Feng, Z.; Gilliland, G. L.; Iype, L.; Jain, S.; Fagan, P.; Marvin, J.; Padilla, D.; Ravichandran, V.; Schneider, B.; Thanki, N.; Weissig, H.; Westbrook, J. D.; Zardecki, C., The Protein Data Bank. Acta Crystallogr., Sect. D: Biol. Crystallogr. 2002, 58, 899-907. 47. Harrison, C. B.; Schulten, K., Quantum and classical dynamics simulations of ATP hydrolysis in solution. J. Chem. Theory Comput. 2012, 8, 2328–2335. 48. Kamerlin, S. C.; Warshel, A., On the energetics of ATP hydrolysis in solution. J Phys Chem B 2009, 113, 15692-8. 49. Akola, J.; Jones, R. O., ATP hydrolysis in water - a density functional study. J. Phys. Chem. B 2003, 107, 11774-11783. 50. Wang, C.; Huang, W.; Liao, J.-L., QM/MM Investigation of ATP Hydrolysis in Aqueous Solution. J. Phys. Chem. B 2015, 119, 3720−3726. ACKNOWLEDGEMENTS This work was supported by funds from Academia Sinica and MOST (Grant # 98-2113-M001-011), Taiwan. T.D. was supported by the project Materials Networking H2020-TWINN2015 and the Institute of Biomedical Sciences, Academia Sinica. The NMR spectra were measured at the High Field NMR Center in Academia Sinica, and at the Instrumentation Center, National Taiwan University. We thank Ms Shou-Ling Huang of the Department of Chemistry, National Taiwan University for assisting with NMR data collection. Authors Contributions The first two authors contribute equally to this work. Notes. The authors declare no competing financial interests.


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How native and non-native cations bind and modulate the properties

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