A water soluble calcium–sodium based coordination polymer: selective release of calcium at specific binding sites on proteins

Abstract

A calcium–sodium based water soluble coordination complex [{Ca₄Na(EGTA)₂(H₂O)₁₃}_n·NO₃] (EGTA = ethylene bis(oxyethylenenitrilo)tetraaceticacid), henceforth named Ca/Na-1, has been synthesized hydrothermally and characterized using spectroscopic and single crystal X-ray diffraction techniques. Single crystal X-ray diffraction of the assembly affirms a two dimensional self assembled net-like supramolecular structure. The complex serves as a biological mimic of a calcium buffer and dispenses felicitous amounts of Ca²⁺ ions at the binding sites on proteins, as shown by SEM imaging and MALDI-TOF spectroscopy. The thermodynamics of binding has also been measured by isothermal titration calorimetry. Protein conformational changes have been characterized by NMR spectroscopy.

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Over the past few decades, coordination polymers have gained attention due to their structural richness and promising applications in supramolecular chemistry, catalysis, drug delivery, gas storage, separation and sensors.¹ Careful selection of metal ion connectors like Zn, Cu, Mn, Ca, Pd, Pt, Fe and Cd into polydentate bridging ligands containing N and O donors has facilitated the evolution of coordination polymers with versatile functionality.² Multi-carboxylic acids are mostly used as linkers for assembly, owing to the variety of coordination modes for the formation of diverse multidimensional architectures.³ Despite the large diversity, utility of coordination polymers is limited in biomedical applications due to their insolubility, toxicity, degradability and lack of suitable techniques for their functional detection. In this context, recent studies have proposed a suitable combination of nontoxic and biocompatible cations, such as Na⁺, Zn²⁺, Fe²⁺ and Ca²⁺, which couple directly with functionalized and therapeutically active linkers in the construction of coordination polymers.^1,4 For the drug development approach, tremendous efforts have led to the design of molecules to encapsulate calcium, and more importantly, to capture and release calcium. Within the cell milieu, free calcium is excluded from the cytoplasm since Ca²⁺ binds less tightly to water and precipitates various cellular phosphates. This exclusion is accomplished by specialized Ca²⁺ binding proteins (CaBPs) whose binding affinities for Ca²⁺ range from nM to mM.⁵ Here, the active site carboxyl and carbonyl groups of the CaBPs serve as chelating centres and form coordination spheres with Ca²⁺. Several 1,2-bis(o-aminophenoxy)ethoxy-N,N,N′,N′-tetraacetic acid (BAPTA)-based photo-induced Ca²⁺ chelators have also been proposed for selective triggering and mapping of Ca²⁺ inside cells.⁵ Though coordination polymers have been exploited in almost every field of research, coordination polymer–protein interactions and metal ion release are still unexplored. In this context, we present a rational design for the incorporation of Ca and Na into an EGTA framework for the selective release and targeted delivery of Ca at designated sites on proteins.

The ligand EGTA was selected owing to its biodegradable and flexible framework with a variety of coordination modes, which are anticipated to provide a multidimensional architecture.⁶ It has also been established that EGTA strongly binds Ca²⁺ in 1 : 1 stoichiometry with a dissociation constant (k_d) in the order of nM, and can mimic the intracellular environment.⁷ In this backdrop, we designed a strategy to alter the stoichiometry of calcium per molecule of EGTA, to increase the k_d of the Ca–EGTA complexation. This can be achieved through the exploitation of the flexible bidentate arms of EGTA to anchor additional metal ions. An optimal reduction in the affinity is expected to confer Ca²⁺ releasing ability to EGTA in the presence of ligands with a higher affinity for Ca²⁺ ions.

Synthesis of the proposed complex, [{Ca₄Na(EGTA)₂(H₂O)₁₃}_n·NO₃], was carried out hydrothermally by reacting Ca(NO₃)₂·4H₂O with EGTA in the presence of NaOH (the detailed procedure is described in the ESI, S1†). Single crystal X-ray diffraction suggests that the structure of Ca/Na-1 crystallizes in a monoclinic crystal system with a space group P2₁/c as shown in Fig. 1A. Data collection and structure refinement parameters, and bond length and bond angle data are shown in the ESI, Tables 1 and 2 (S2 & S3).† An asymmetric unit of each EGTA anion acts as an octadentate ligand connecting four metal centers (three Ca(II) ions and one Na ion) and forms a cage-like structure with co-crystallized nitrate molecules. Two Ca(II) ions (Ca1 and Ca3) are equatorially connected in an octa-coordinated fashion and bind six oxygen atoms from the carboxylate groups and two nitrogen atoms belonging to the same EGTA ligand, with the typical Ca–O bond length values ranging from 2.363(5) to 2.592(5) Å, which fall in the reported range.⁸ Two Ca(II) centers coordinated with carboxylate dianions of EGTA are bridged by other Ca(II) and Na(I) ions and are separated by a distance of 8.179 Å. In this way, the two EGTA⁴⁻ ligands link the metal ions acting as building blocks and form a cage with a 13-membered metallacycle. Overall, thirteen disordered coordinated water molecules are present in Ca/Na-1. Furthermore, Ca/Na-1 is interconnected and provides an extended 2D net-like supramolecular framework, as depicted in Fig. 1B. The solvent accessible volume calculated by PLATON analysis⁹ is 6013 ų, which is 14.5% of the unit cell volume.

Fig. 1 (A) A cage-like asymmetric unit of Ca/Na-1 (hydrogen omitted for clarity). (B) The 2D mesh-like structure of Ca/Na-1.

A characteristic peak of the υ_as(–COO–) vibration of the free ligand observed at 1744 cm⁻¹ is shifted to 1635 cm⁻¹ in the infrared spectrum of Ca/Na-1. The υ_s(COO–) is at 1384 cm⁻¹ and blue-shifts 17 cm⁻¹, compared to that of EGTA (1401 cm⁻¹), supporting the coordination of the deprotonated ligand to the metal ions in the complex.¹⁰ MALDI-TOF mass spectrometry of Ca/Na-1, recorded in Tris–HCl buffer (pH 7.4) and 100 mM NaCl, showed a peak at 1232.19 corresponding to its parent molecular ion (ESI Fig. 1 and S4†). The higher polymers could not be detected. The absence of larger fragments may be due to the rupture of the metal–ligand bond under the conditions applied.¹¹ However, the isolation of the same complex from its solution in water supported the intactness of Ca/Na-1 even after dissolution in water. The UV-visible absorption and emission spectra are shown in the ESI, Fig. 2 (S5).† The electronic absorption spectrum shows peaks at λ_max 302, 354 and 388 nm. The first two peaks are assigned to π–π* transitions and the latter peak arises from n–π* transition respectively. An intense emission peak is displayed at λ_max 452 nm on excitation at λ_max 388 nm. The emission arises from the ligand centre.¹² To check the stoichiometric effect on the structure, UV-vis titration of 10 mM EGTA solution was carried out with different equivalents of Ca(NO₃)₂·4H₂O in the presence of 40 mM NaOH (ESI Fig. 3 (S6)†). No significant changes in the spectral features were observed, confirming that the observed stoichiometry is almost stable. The 1D-¹H, ¹³C and ²³Na NMR spectra of Ca/Na-1 were recorded in Tris–HCl buffer (pH 7.4) with 10% D₂O (ESI Fig. 4 (S7)†). The combined chemical shifts for the ¹H and ¹³C spectra are listed in the ESI, Table 3 (S8).† The peaks at 180.41, 69.14, 68.18, 61.82 and 57.56 ppm correspond to the carboxy groups (–CO₂), the CH₂ groups of (–NCH₂CO₂), the two types of CH₂ groups of (–O–CH₂–CH₂), and the CH₂ groups of acetates (–NCH₂CH₂–), respectively. A shift of δ 0.967 ppm, corresponding to a single peak of ²³Na compared to standard NaOH, indicated the presence of Na in the Ca/Na-1 complex. Incorporation of Ca into the coordination polymer was indirectly verified, since ⁴⁰Ca was used for synthesis (as ⁴⁰Ca(NO₃)₂·4H₂O, >99% pure).

As described earlier, it could be hypothesized that molecular asymmetry might result in the lowering of binding affinity of one or more Ca ions in Ca/Na-1. To test this hypothesis, Ca/Na-1 was titrated against a β/γ-crystallin protein, M-crystallin. This protein has two asymmetric motifs that bind Ca²⁺ ions with moderate and low binding affinities respectively. Also, it undergoes subtle conformational changes upon binding to Ca²⁺.¹³ An overlay of the sensitivity enhanced 2D-[¹⁵N–¹H] heteronuclear single quantum correlation (HSQC) spectra of the protein in the presence and absence of Ca/Na-1 showed perturbations in amide proton chemical shifts (Fig. 2). Ca²⁺ binds to M-crystallin with a pentagonal bipyramidal geometry at site 1 and an octahedral geometry at site 2 along with three coordinating water molecules.¹⁴ For reference, the spectra were recorded using standard CaCl₂ as the titrant (ESI Fig. 5 (S9)†). The results corroborate the release of Ca²⁺ by Ca/Na-1 during titration with concomitant conformational changes in the protein. However, no significant change occurs in the ²³Na NMR spectrum of the Ca/Na-1 complex (Fig. 3A) in the presence of the protein, supporting that Na⁺ ions remain intact during the release of Ca²⁺ ions.

Fig. 2 NMR titration of Ca/Na-1 with M-crystallin. Overlay of sensitivity enhanced [¹H–¹⁵N] HSQC spectra of the free protein (blue) and the Ca/Na-1 bound protein (red). The spectra were recorded at 25 °C in 25 mM Tris–HCl buffer (pH 7.4) and 100 mM NaCl.

Fig. 3 (A) Overlay of the ²³Na spectra of pure Ca/Na-1, NaCl standard and M-crystallin protein + pure Ca/Na-1 solution (B) ITC thermogram of Ca/Na-1 titration with M-crystallin. The protein concentration was 60 μM in 25 mM Tris–HCl buffer, pH 7.4 and 100 mM NaCl.

The extent of interaction of Ca/Na-1 with M-crystallin and the binding energetics were estimated from an isothermal titration calorimetry (ITC) profile. The overall dissociation constant (k_d) is 52 μM and is best fitted to a two-site sequential binding model (Fig. 3B). The individual dissociation constants were 19 μM and 147 μM and binding of Ca²⁺ at both the sites was enthalpically and entropically favored (ESI Table 4 and S10†). For comparison, ITC experiments repeated under identical conditions using 1 mM CaCl₂ yielded a k_d of 82.3 μM, consistent with the reported value.¹³ The most plausible rationale for the release of free Ca²⁺ from Ca/Na-1 can be deduced from the fact that the activation enthalpy and entropy (ΔH and ΔS) for Ca²⁺ exchange between EGTA and M-crystallin are distinct and are in favor of the protein. Also, the decreased binding affinity of Ca/Na-1 to Ca²⁺ can be ascribed to the asymmetry of the metal centers in the complex. In this context, it is noteworthy to mention the difference in thermodynamic parameters, which can be attributed to an anion effect in the titrant (OH⁻ in case of Ca/Na-1 and Cl⁻ and OH⁻ in case of CaCl₂·2H₂O).¹⁵

The stoichiometry of binding was estimated from a suite of NMR titrations carried out using different protein : Ca/Na-1 concentrations (1 : 0.5, 1 : 1, 1 : 2 and 1 : 3). Saturation of both the Ca²⁺ binding sites was observed with a 1 : 3 protein to Ca/Na-1 ratio. It is speculated that Ca4 is dislodged from Ca/Na-1 due to the higher energy of Ca[H₂O]₄²⁺·2H₂O in the coordination sphere.¹⁶ Thereby, 2 Ca²⁺ are released per 3 molecules in order to saturate the binding sites on the protein. The MALDI-TOF mass spectrum of the protein with Ca/Na-1 in a 1 : 2 ratio showed [M + H]⁺ at m/z 9259.12 and 9390.81, which correspond to the apo and holo protein respectively (Fig. 4A). It was also observed that the bond length and bond angle parameters contributed to the release of Ca²⁺ ions. The bond lengths of Ca(4)–O(26), Ca(4)–O(11), Ca(4)–O(12), Ca(4)–O(7) (2.510–2.560 Å) were found to be larger compared to other Ca–O bond lengths (2.359–2.487 Å). This verified that the larger bond length of the Ca(4)–O bond per unit composition of the complex facilitated its release, as depicted in Fig. 1A. Furthermore, the smaller bond angle of O(27)–Ca(4)–O(11) contributes to the spontaneous release of Ca²⁺.

Fig. 4 (A) A representative MALDI-TOF mass spectrum of M-crystallin titration with Ca/Na-1 at a 1 : 2 ratio of protein vs. Ca/Na-1. (B) A FE-SEM image of Ca/Na-1 after Ca²⁺ release. (C) A schematic showing Ca²⁺ release from Ca/Na-1 and the conversion of apo M-crystallin to the holo form.

A mass difference of 131.69 supports the chelation of two Ca²⁺ ions along with three water molecules. Post binding, Ca/Na-1 undergoes polymerization to form branched, porous nanorods with an average length of ∼3 μm and a mean diameter of ∼200 nm, as observed in the FE-SEM imaging (Fig. 4B). The M-crystallin–Ca/Na-1 complexation is schematically illustrated in Fig. 4C. The nanorods thus formed disintegrate within ∼48 h, ensuring degradability in physiological conditions.

The results demonstrate the utility of the calcium–sodium based coordination polymer which mimics the role of a Ca²⁺ buffer in living cells, as corollary to Na⁺/Ca²⁺ exchange proteins (NCX) that act in a reversible fashion to maintain concentration gradients of individual ions across membranes. NCX bind to Ca²⁺ with low affinity but can transport as many as 5000 ions per second, making them fast dispensers of the ion during the generation of action potentials for nerve impulses or signal transduction.¹⁷ Disruptions in the functioning of these proteins and subsequent low blood Ca²⁺ levels lead to adverse effects including neurological impairment, seizures, abnormal heart rhythms and certain cancers.¹⁸ In such cases, developing simple strategies to replenish the Ca²⁺ levels becomes indispensible for averting any complications.

In summary, we propose that the EGTA based coordination polymer forms an apt system to incorporate biologically relevant metal ions for targeted metal ion delivery to proteins. In terms of synthesis, Ca/Na-1 is obtained in aqueous solutions, instead of organic solvents. In this particular case, Ca/Na-1, EGTA serves as a biological mimic of a Ca²⁺ buffer. In addition to Ca²⁺ ion chelation by EGTA, Ca/Na-1 seems to release felicitous amounts of Ca²⁺ ions at designated target sites on proteins. Thus, Ca/Na-1 can be readily deployed to sequester Ca²⁺ for selective manipulation of cellular Ca²⁺ levels and associated physiological functions.

Acknowledgements

RG and LM thank CSIR, New Delhi, India for financial assistance. AS thanks ICMR, India for the SRF. Mr Rudheer Bapat is acknowledged for helping with the FE-SEM imaging.

References

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Footnotes

† Electronic supplementary information (ESI) available: Detailed experimental procedures include synthesis, crystallographic data, bond lengths, bond angle data, MALDI-TOF mass spectrum, NMR spectra, UV-vis and emission spectra of Ca/Na-1, comparison of M-crystallin titration using Ca/Na-1 and CaCl₂ as the titrant, and thermodynamic parameters of M-crystallin-Ca/Na-1 complexation. CCDC 981423. For ESI and crystallographic data in CIF or other electronic format see DOI: 10.1039/c4ra01005a

‡ These authors contributed equally.

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