Molecular Recognition Study on the Binding of Calcium to Calbindin

Article pubs.acs.org/JPCB

Molecular Recognition Study on the Binding of Calcium to Calbindin D9k Based on 3D Reference Interaction Site Model Theory Yasuomi Kiyota and Mayuko Takeda-Shitaka* School of Pharmacy, Kitasato University, 5-9-1 Shirokane, Minato-ku, Tokyo 108-8641, Japan ABSTRACT: Ca2+-binding proteins are widely distributed throughout cells and play various important roles. Calbindin D9k is a member of the EF-hand Ca2+-binding protein family. In this study, we examined the binding of Ca2+ to calbindin D9k in terms of the free energy of solvation, as obtained by 3D reference interaction site model theory, which describes the statistical mechanics of liquids. We also investigated the main structural biological factor using spatial decomposition analysis in which the solvation free energy values are decomposed into the residue. We found some characteristic residues that contribute to stabilization of the holo-structure (Ca2+binding structure). These results indicated that, in the holo-structure, these residues are newly exposed to solvent. Subsequently, the gain in solvation free energy, involving a conformational change and exposure to solvent, forms the driving force for binding of the Ca2+ ion to the EF-hand.

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INTRODUCTION As an intracellular signaling ion, Ca2+ is involved in an array of cellular functions.1,2 Ca2+-binding proteins (CaBPs) are involved in intra- and extracellular processes, as well as in Ca2+ uptake, transport, and homeostasis. These proteins are characterized by a common helix−loop−helix ion-binding motif, termed an EF-hand, which binds divalent metal ions. EF-hand motifs are similar in their amino acid sequences and structure, although CaBPs perform a diverse range of biological functions. These functions can be divided into two classes: one is Ca2+ sensors, which transduce Ca2+ signals, and the other is Ca2+ signal modulators, which modulate the waveform of the Ca2+ signals or participate in Ca2+ homeostasis. Ca2+-buffering proteins make up a smaller subset of the CaBP family. Calbindin D9k, which is a CaBP, is involved in intracellular buffering of Ca2+ and/or uptake of Ca2+ from the intestinal brush border membrane and its transport to the membrane.3 In terms of modulation of Ca2+ signaling, calbindin D9k can help to control the waveform of Ca2+ signaling both spatially and temporally by binding free Ca2+. Calbindin D9k also has the ability to remove the potentially harmful ion from the cytoplasm. However, the in vivo significance of this protein to pathological conditions remains uncertain, and continues to be investigated, along with that of other S100 subgroups, which have garnered attention in the medical field in recent years.4,5 This protein has been selected as an initial target and primary model system for various experimental and theoretical studies, because of its suitable size (75 residues) and stability over a wide range of experimental conditions, including X-ray crystallography and nuclear magnetic resonance (NMR) spectroscopy; it has also been used in theoretical simulation studies. Numerous high-resolution X-ray crystallographic studies of this superfamily have indicated that the basic structural domains © 2014 American Chemical Society

in these proteins are pairs of EF-hands that are connected by loops of varying length in the holo-state or doubly loaded [(Ca2+)2] state.6 Calbindin D9k is comprised of two EF-hands with a 14-residue noncanonical loop in the N-terminal region; this region of the EF-hand is termed “EF1”, while “EF2” refers to the typical 12-residue canonical loop in the C-terminal region (Figure 1). Although calbindin D 9k has been characterized by extensive biophysical and thermodynamical techniques, such as X-ray diffraction, these structures have yielded little insight into the conformational response to Ca2+ binding, due to the absence of observed structural changes between the apo-state and Ca2+-bound state.7 Yet, this is important, because the conformational response following ion binding is usually regarded as a key factor determining protein activity. NMR and restrained molecular dynamics (MD) studies have been used to investigate differences in the three-dimensional (3D) structure of calbindin D9k in solution between the apostate and the holo-state, in terms of the changes induced by Ca2+ binding to the EF-hand. These studies provide a basis for understanding the correlation between conformational responses and activity.8,9 The conformation of the apo-state has been determined by the distance obtained from NMR and restrained MD simulation, and that of the holo-state was obtained by using MD simulation based on X-ray structures.10 A detailed comparison of both structures has suggested that Ca2+ binding does not induce a large conformational change in the EF-hand motif in calbindin D9k, unlike that which occurs in calmodulin and troponin C, where binding leads to the activation or inactivation of the protein. Received: May 16, 2014 Revised: September 12, 2014 Published: September 24, 2014 11496

dx.doi.org/10.1021/jp504822r | J. Phys. Chem. B 2014, 118, 11496−11503

The Journal of Physical Chemistry B

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If accurate solvation free energy of the protein can be obtained, decomposition of the physical quantity into a set of fundamental units like amino acids is important for understanding the binding mechanism and the key factors involved. In fact, decomposition into the basic units is an essential strategy in theoretical studies of proteins.19,20 Spatial decomposition analysis (SDA) of biomolecules, based on the 3D-RISM theory of molecular liquids, was originally proposed by Yamazaki et al.,21 founded on 3D integral equation theory. The 3D correlation functions obtained by 3D-RISM theory reveal the 3D-spatial information around the solute, which is equal to that obtained by mapping of the thermodynamic quantities, such as the solvation free energy. The essential factor in this analysis is that the thermodynamic properties of some specific interaction sites are summed from the partial contributions based on the volume elements that are defined by the distance from the interaction sites, which are atoms, functional groups, or residues of the protein. We believe that this decomposition approach can be employed in Ca2+-binding protein systems to understand the contributions of particular functional residues. In this paper, to identify specific interactions within the globular domain of calbindin D9k that govern the magnitude of the conformational response, we obtained structures from the Brookhaven Protein Data Bank (PDB)22 and calculated the distribution functions of the apo-state (PDB ID: 1CLB; NMR of 33 structures)8 and the holo-state (PDB ID: 3ICB; doubly loaded X-ray structure)6 of the protein, including Ca2+, using 3D-RISM theory. Then, in order to analyze the details of its physical properties, we applied the SDA approach to each amino acid.

Figure 1. Schematic structure of calbindin D9k with two bound Ca2+ ions inside EF-hands. The most important structural elements of calbindin D9k are two EF-hands, which are depicted in red. A 14residue noncanonical loop in the N-terminal site, we defined this section of the EF-hand as “EF1”, and a typical 12-residue canonical loop in the C-terminal site was defined as “EF2”. The Ca2+ ion in the X-ray structure is shown as a yellow-colored sphere. This figure was drawn using PyMOL (http://www.pymol.org).

Although expensive in terms of computational cost, molecular simulations at an equilibrium state can obtain microscopic solute and solvent structures and are considered to be the most accurate modeling approach for studying proteins in solution. However, theoretical prediction of ion binding by a protein is not trivial, even in MD simulation. In the present paper, we employed a 3D molecular theory of solvation, also known as the 3D reference interaction site model (3D-RISM) theory,11,12 to obtain 3D information on the thermodynamic properties of calbindin D9k. The 3D-RISM theory is a useful and powerful tool for studying solvation thermodynamics of macromolecules in various environments and has been proven to describe the thermodynamics and properties of a solvation structure successfully.13−16 For accurate and quantitative studies of the Ca2+-binding protein, it is important to assess the long-range electrostatic interactions between not only the ion and the protein but also between ions. For instance, the Ca2+-to-Ca2+ distance in the holo-state of calbindin D9k is on the order of 12 Å. Therefore, truncation of electrostatic interactions around 8−10 Å as a default setting in MD simulation implies that the Ca2+-to-Ca2+ repulsion (which is on the order of +110 kcal/mol) can be ignored.17 Moreover, it is difficult to represent the influence of the surrounding bulk solvent caused by periodic boundary conditions or a finite droplet of water molecules realistically. For example, the hydration free energy of Ca2+ (−381 kcal/mol in bulk water) is underestimated by more than 30 kcal/mol with a 20 Å radius according to the Born model of solvation.10 As mention above, in view of the modest changes in structure and free energy during Ca2+ binding, the 3D-RISM method is appropriate for use in this system. When using MD simulation, it is required that the balance between avoiding truncation for evaluating long-range interactions and decreasing computational time with a cutoff is maintained; however, when using the 3D-RISM theory, consistent electrostatics are essential.18

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METHODS Statistical Mechanics of Solution According to the 3D-RISM Theory. In this study, we can consider the system composed of calbindin D9k molecules and calcium chloride solution as the species. In this section, we provide only a brief outline of the 3D-RISM theory, because it is described elsewhere in more detail.23 The 3D-RISM theory originates from the Ornstein−Zernike (OZ) integral equation24 for a liquid mixture. The equation is written as hij(12) = c ij(12) +

∑ ρl ∫ d(3) c il(13) hlj(32) l

(1)

where the superscripts denote the species in the mixture. h(12) and c(12) denote the total and direct correlation functions, respectively. ρl is the number density of the solution species l. In the statistical mechanics of liquid, the solution species fall into solvent and solute, which are not treated equivalently. Solvent has enough number in solution, and then there are interactions between each solvent species like water or ions. Solute is under infinite dilution (ρl = 0), and then there is only one molecule in the system, such as protein. Accordingly, the OZ equation (eq 1) is rewritten for considering the solvent around the protein as the solute (u)−solvent (v) equation (eq 2). huv (12) = c uv(12) + ρ v

∫ d(3) c vv(13) hvu(32)

(2)

The 3D-RISM integral equation for solvent around the protein can be derived from the solute−solvent OZ equation (eq 2) with averaging over the orientations of solvent sites around the solute site. This partial averaging by definition 11497



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parameter set29 was adopted as potential parameters for the protein. We adopted the Lorentz−Verthelot combination rule to combine the Lennard−Jones parameters. Information on the susceptibility of the pure solvent, which is given by χγ′γ(r) = ωγ′γ(r) + ργ′hγ′γ(r), can be separated into the 2 intramolecular distribution function ωγ′γ(r) = δ(r − lγ′γ)/4πlγ′γ , which specifies the geometry of solvent molecules with site− site distance, lγ′γ, and the intermolecular site−site total correlation function, hγ′γ(r), times ργ. The radial correlations, hγ′γ(r), of pure solvent are obtained, in advance of the 3DRISM calculation, from the one-dimentional (1D) RISM integral equation theory for solvent−solvent systems. To solve this system, we prepared an aqueous solution of 0.01 mol/kg CaCl2, which is close to the experimental conditions, and performed calculations at 298 K under normal pressure. In this study, we employed the dielectrically consistent RISM method, which requires an experimental value for the dielectric constant30 and then employs the dielectric constant for pure water, viz., 78.5. SDA Based on 3D-RISM Theory. To analyze the thermodynamic properties of residues, a basic concept used in SDA with the 3D-RISM method is to classify the 3D solvation free energy in the region defined by each residue.21 Following from the 3D-RISM equations (eq 4) and KH closure (eq 5), the solvation free energy, μ, is derived similarly to the Singer−Chandler expression.31 In the expression, the formula is in the 3D form and is modified in the h2 term.

reduces the solute−solvent 6D correlation functions, such as huv(12) ≡ huv(r12, Ω1, Ω2), to the 3D correlation functions of solvent interaction sites γ around the solute molecule, hγuv (r) ≡ hγuv (r1γ , Ω1) =

1 Ω

∫ dΩ2 hγuv(|r1γ − r2γ|, Ω1, Ω2) (3)

where r1γ = rγ − r1 is the intermolecular vector from the solute to solvent site γ. The solvent−solvent correlation functions are also averaged over orientations that fix the distance between the interaction sites and are reduced to 1D correlation functions. After this treatment, the 3D-RISM integral equation25,26 is derived as hγ (r) =

∑ ∫ d r′cγ′(r′) χγγ′ (|r − r′|) (4)

γ′

.Then, the 3D-RISM integral equation is coupled with the Kovalenko−Hirata (KH) closure approximation,26−28 which has been proven to be appropriate for describing complex liquids and solutions and various biomolecular systems ⎧ ⎪ exp(d γ (r)) for d γ (r) ≤ 0 gγ (r) = ⎨ ⎪ ⎩ 1 + dγ(r) for dγ(r) > 0

(5a)

dγ(r) = −uγ (r)/kBT + hγ (r) − cγ(r)

(5b)

where hγ(r) is related to the 3D distribution function gγ(r) = hγ(r) + 1, which gives the normalized probability of finding the interaction site γ of solvent molecules at position r around the solute molecule, such as a protein. As mentioned above, cγ(r) has the asymptotics of the solute−solvent site interaction potential: cγ(r) ≈ −uγ(r)/kBT for superposition approximation, and kBT is the Boltzmann constant multiplied by the temperature of the solution. The interaction potential, uγ(r), is described as the sum of the electrostatic interaction and Lennard−Jones (LJ) potential as follows: uγ (r) =

∑ a

μ = kBT ∑ ργ

-(r) =

(7a)

1 2 1 hγ (r) Θ( −hγ (r)) − cγ(r) − hγ (r) cγ(r) 2 2

(7b)

where Θ(x) is the Heaviside step function ⎧1 for x ≥ 0 Θ(x) = ⎨ ⎩ 0 for x < 0

12 ⎧ σaγ ⎞ ⎪⎛ + ∑ 4εaγ ⎨⎜ ⎟ ⎪ |r − r | |r − ra| a ⎠ a ⎩⎝

qaqγ

⎛ σaγ ⎞6 ⎫ ⎪ −⎜ ⎟⎬ ⎝ |r − ra| ⎠ ⎪ ⎭

We represent 3D mapping of the solvation free energy around calbindin D9k, as shown in Figure 2a. As stated in the paper by Yamazaki and Kovalenko,21 the 3D grid points, which are used to solve the 3D-RISM equations, are classified into the region based on Voronoi tessellation,32,33 as shown in Figure 2b. A basic concept of this classification is that “a grid point inside the excluded volume is related to the LJ sphere with the center closest to the grid point, and the grid points outside the LJ sphere region are partitioned based on the radical plane in which a grid point is related to the LJ sphere with the surface closest to the grid point.” In Figure 2b, each colored region in the box corresponds to a part of protein with the same color. On the basis of the above scheme, the solvation free energy of atom i (μatom ) can be calculated by integrating the correlation i function, and these small components of solvation free energy are attributed to atom i as

(6)

where qa denotes a partial charge on site a. σ and ε are the LJ parameters with the usual meanings. Table 1 summarizes the potential parameters and structural information that is used in these calculations. In the 3D-RISM calculation, the Amber99 Table 1. Summary of Lennard−Jones Potential Parameters of Solvent Species and Structure of Solvent Water O of watera H of watera Ca2+ b Cl− O−H (Å) ∠HOH (deg)

∫ dr -(r)

γ

σ (Å)

ε (kcal/mol)

q (e)

3.166 0.400 2.412 4.417 1.000 109.47

0.1550 0.0460 0.4497 0.1178

−0.82 0.41 2.00 −1.00

μiatom = kBT ∑ ργ γ

∫V ∈atom i dr -(r)

(8)

a

Parameter set for water employed the simple point charge (SPC) model.43 bParameter for Ca2+ ion is found in the optimized potential for the liquid state (OPLS) parameter set.44,45

and the solvation free energy of residue X (μres X ) can be added from the atom component as 11498



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protein, because the 3D-RISM calculations are performed with each conformation of the apo- and holo-state of calbindin D9k.

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RESULTS AND DISCUSSION Comparison between Calculated Thermodynamic Properties and Experimental Data. First, 3D-RISM calculations were carried out for the holo-state [doubly loaded; (Ca2+)2] in aqueous solutions of 0.01 mol/kg CaCl2. The structure of the holo-state was prepared from PDB (ID: 3ICB).6 In order to apply the 3D-RISM theory, all heteroatoms (water and ions) were removed from the structures. In Figure 3, the 3D distribution function of Ca2+ at the protein surface is shown. Clear distribution was observed around EF-hands, which implies that Ca2+ ions were bound to each EF-hand. In the figure, the distribution function, g(r), is shown in yellow when >75; g(r) > 75 denotes that the probability of finding

Figure 2. Three-dimensional (3D) mapping of solvation free energy and Voronoi tessellation. (a) Positive portion of solvation free energy is shown as a white surface (μsolv ≥ 10 kcal/mol) and the negative portion is shown in black (μsolv ≤ −10 kcal/mol). Solvation free energy was calculated using the Singer−Chandler expression (eq 5), based on the obtained 3D distribution function. (b) The 3D grid points are attributed to each atom by Voronoi tessellation. Each colored region in the box corresponds to a part of protein with the same color. on residue X

μ Xres =

∑

μiatom

Figure 3. Three-dimensional (3D) distribution functions obtained by 3D-RISM calculations. 3D distribution functions of oxygen of the Ca2+ ion and the oxygen of water are shown as yellow and cyan surfaces, respectively. Additionally, the Ca2+ ion and the oxygen of water in the X-ray structure are shown as yellow and blue colored transparent spheres, respectively. (a) 3D distribution functions around calbindin D9k. (b) An enlarged view of the 3D distribution functions around EF1. (c) An enlarged view of the 3D distribution functions around EF2. Criteria for 3D distribution functions are 75 [gCa2+(r) > 75] for the Ca2+ ion and 8.0 [gO(r) > 8.0] for the oxygen of water.

i

= kBT ∑ ργ γ

∫V ∈residue X dr -(r)

(9)

Therefore, the sum of μres over all the residues is equal to the total solvation free energy μ calculated by using eqs 7a and 7b. Note that μres is dependent on a given conformation of the 11499



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Ca2+ ions at the particular position is 75 times greater than that in the bulk of the protein. A value of 75 is significantly higher than that for 3D-RISM calculations of other CaBPs. For instance, the highest peak of phospholipase A2 (PLA2), which can also bind Ca2+, is about 40,34 due to the difference in the number of negatively charged residues. In calbindin D9k and canonical EF-hands, at least five residues are related to Ca2+ coordination, whereas in PLA2 only four, which involve carboxyl groups, are involved. Figure 3b,c shows an expanded view of the water and Ca2+ distributions around residues in the EF-hands in calbindin D9k. The structure of heteroatoms according to X-ray crystallography is also shown for comparison. Not only the Ca2+ ion but also the oxygen in water is distributed around the EF-hands. In particular, the position of water, which is coordinated to the Ca2+ ion, is represented for each EF-hand. The results indicated that Ca2+ binding is strongly related to water binding, supplementing the coordination number of the Ca2+ ion. It is straightforward to obtain the 1D distribution, or the radial distribution function (RDF). The RDFs can be obtained by averaging the 3D-distribution function over a given direction around a specified center.18 ga1D(r, r0) =

1 4π

∫ ga(r + r0) dr ̂

(10)

where r̂ is the direction of vector r and r0 indicates a center for the averaging. In this paper, the locations of two calcium ions in the X-ray structure of the holo-form were chosen as the averaging center. In Figure 4a,b, the RDFs of the Ca2+ ion are shown. The RDF of the Ca2+ peaks at the center of its atom in the X-ray structure. We reproduced binding of two Ca2+ ions by calbindin D9k with high affinity. The higher peak of EF2 compared to that of EF1 indicates that the distribution of the Ca2+ ion obtained by means of 3D-RISM was in good agreement with experimental data.35 Second, we performed 3D-RISM calculations for the apostate structure in the same solution. The experimental structural studies showed that calbindin D9k does not undergo any major conformational changes upon binding either one or two Ca2+ ions. The rearrangements of the side chains upon Ca2+ binding to EF2 did not affect the backbone conformation of EF1 significantly and induced only modest structural changes in the nonpolar side chains. The fluctuations of calbindin D9k in each state were similar, and various NMR EF-hand structures in a conformation-independent frame incorporate structural fluctuations from the apo- to holo-states [(Ca2+)1 and (Ca2+)2] under the same physiological conditions. Therefore, the additional structures were prepared from PDB code 1CLB,8 which includes 33 independent structures. We obtained the solvation energy (μsolv), which can be calculated by eqs 7a and 7b for each frame and the structural potential energy of the protein (Econf). The total energy (Etot) of the system must be the sum of these, unless electron transfer occurs. Figure 5 shows the results of the analysis for Etot of each frame. The total energy of each state is constant, averaging −1250.9 kcal/mol (correlation coefficient between solvation energy and potential energy of the protein is −0.964). This behavior of Etot indicates that these frames are apo-structures in a state of thermal equilibrium. In order to include the Ca2+ ions in the solvent, the holo-state can be taken from the frame with a higher solvation energy, because the partial EF-hand had already been constructed in the frame. In fact, the maximum value of the Ca2+ peak correlated with the solvation free energy.

Figure 4. Radial distribution functions obtained by averaging the three-dimensional (3D) distribution functions. Radial distribution functions of the Ca2+ ion around binding sites: (a) EF1 and (b) EF2. The center of the Ca2+ ion in the X-ray structure was chosen as the origin for averaging the 3D distribution functions.

Figure 5. Plots of total energy from apo-structures. Total energy of one conformation is calculated by summation of the potential energy of the protein (Econf, open) and solvation free energy (μsolv, shaded) for each structure.

Results of Spatial Decomposition Analysis. In order to obtain structural biological information, we carried out SDA based on 3D-RISM calculations. We dealt with one structure as a holo-state, because we considered that the structural fluctuation in the holo-structure is smaller than that in the apo-structures. Indeed, some experimental data36 support this 11500



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Figure 6. SDA of the holo-structure of calbindin D9k. Results of SDA for the solvation free energy μres of each residue. Hatched regions show the sequence of each EF-hand. The arrows indicate the residues that are directly related to Ca2+ coordination.

Figure 7. SDA differences between holo- and apo-structures. SDA differences for the solvation free energy μres of each residue. The lines within the boxes indicate the median values of the data. The hatched regions show the sequences of each EF-hand. The arrows indicate the residues that contribute to stabilization of the holo-structure against all of the apo-structures.

hypothesis. The values of the solvation free energy μ of the holo-state (PDB ID: 3ICB6) were decomposed by SDA into the partial contributions of the residue, μres, as shown in Figure 6. The hatched region indicates the EF-hands, and the residues that are directly involved in Ca2+ coordination, such as Asp or Glu, exhibited stabilization by solvation with the Ca2+ ion. Only Gln22 demonstrated different characteristics as compared with other coordinated residues, because the side chain of the residue is directed away from the Ca2+ ion. We also subsequently applied SDA to the apo-structures (PDB ID: 1CLB8) and obtained differences in energy between the apoand holo-states (Figure 7). Interestingly, some residues that exhibited smaller changes in differential solvation free energies obeyed the position of the amino acid residues in the protein. For instance, hydrophilic residues are found on the protein surface, while hydrophobic residues appear inside the protein. On the other hand, the larger-changed residues could be found inside and outside of the protein.

In Figure 7, some characteristic residues always exhibited negative values; these residues contributed to stabilization of the holo-structure against the 33 apo-structures. Upon observing these structures, it became clear that these residues shared a common feature in terms of structural biology (Figure 8). They included two types of residues: one was residues located at the initial residue of the EF hand, viz., Ala14 (EF1) and Asp54 (EF2), while the other was bound to the main chain via Ca2+, viz., Glu17 (EF1) and Glu60 (EF2). This result indicated that, in the apo-structures, the Ca2+-binding site is buried inside the protein. However, in the holo-structure, or in the Ca2+-binding structure, these residues are newly exposed to solvent. The gain of solvation free energy, upon conformational change and exposure to solvent, is converted into a driving force due to binding of the Ca2+ ion at the EF-hand. From the various computational studies using MD simulation or high-resolution structural databases, the carboxylate oxygen atoms of Glu60 seem to be important for binding to Ca2+ by EF1. Recently, a hypothesis has been put forward 11501



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Our present study shows that 3D-RISM is a useful tool for detecting and predicting changes in solvation thermodynamics caused by molecular recognition. Other studies16,18,34 showed that 3D-RISM is a useful tool for protein mutation studies, protein−ligand interaction studies, etc. Taken together, these studies using 3D-RISM indicate that 3D-RISM will be an essential tool for research in the field of life sciences, such as prediction and analysis of structure−function relationships and drug discovery.

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AUTHOR INFORMATION

Corresponding Author

*Phone: +81-3-5791-6330. E-mail: [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS Molecular graphics images were produced with UCSF Chimera40 and gOpenMol.41,42 The authors also thank Prof. F. Hirata (Ritsumeikan University, Kyoto, Japan) for his helpful suggestions.

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REFERENCES

(1) Gifford, J. L.; Walsh, M. P.; Vogel, H. J. Structures and Metal-IonBinding Properties of the Ca2+-Binding Helix−Loop−Helix EF-Hand Motifs. Biochem. J. 2007, 405, 199−221. (2) Berridge, M. J.; Bootman, M. D.; Roderick, H. L. Calcium Signalling: Dynamics, Homeostasis and Remodelling. Nat. Rev. Mol. Cell Biol. 2003, 4, 517−529. (3) Christakos, S.; Gabrielides, C.; Rhoten, W. B. Vitamin DDependent Calcium Binding Proteins: Chemistry, Distribution, Functional Considerations, and Molecular Biology. Endocr. Rev. 1989, 10, 3−26. (4) Santamaria-Kisiel, L.; Rintala-Dempsey, A. C.; Shaw, G. S. Calcium-Dependent and -Independent Interactions of the S100 Protein Family. Biochem. J. 2006, 396, 201−214. (5) Sakatani, S.; Seto-Ohshima, A.; Shinohara, Y.; Yamamoto, Y.; Yamamoto, H.; Itohara, S.; Hirase, H. Neural-Activity-Dependent Release of S100B from Astrocytes Enhances Kainate-Induced Gamma Oscillations in Vivo. J. Neurosci. 2008, 28, 10928−10936. (6) Szebenyi, D. M.; Moffat, K. The Refined Structure of Vitamin DDependent Calcium-Binding Protein from Bovine Intestine. Molecular Details, Ion Binding, and Implications for the Structure of Other Calcium-Binding Proteins. J. Biol. Chem. 1986, 261, 8761−8777. (7) McPhalen, C. A.; Strynadka, N. C. J.; James, M. N. G. CalciumBinding Sites in Proteins: A Structural Perspective. Adv. Protein Chem. 1991, 42, 77−144. (8) Skelton, N. J.; Kördel, J.; Chazin, W. J. Determination of the Solution Structure of Apo Calbindin D9k by NMR Spectroscopy. J. Mol. Biol. 1995, 249, 441−462. (9) Nelson, M. R.; Thulin, E.; Fagan, P. A.; Forsén, S.; Chazin, W. J. The EF-Hand Domain: A Globally Cooperative Structural Unit. Protein Sci. 2002, 11, 198−205. (10) Marchand, S.; Roux, B. Molecular Dynamics Study of Calbindin D9k in the Apo and Singly and Doubly Calcium-Loaded States. Proteins 1998, 33, 265−284. (11) Imai, T.; Kovalenko, A.; Hirata, F. Solvation Thermodynamics of Protein Studies by the 3D-RISM Theory. Chem. Phys. Lett. 2004, 395, 1−6. (12) Imai, T.; Hiraoka, R.; Kovalenko, A.; Hirata, F. Water Molecules in a Protein Cavity Detected by a Statistical−Mechanical Theory. J. Am. Chem. Soc. 2005, 127, 15334−15335. (13) Yoshida, N.; Phongphanphanee, S.; Maruyama, Y.; Imai, T.; Hirata, F. Selective Ion-Binding by Protein Probed with the 3D-RISM Theory. J. Am. Chem. Soc. 2006, 128, 12042−12043.

Figure 8. The holo-structure around the EF-hands of calbindin D9k, showing characteristic residues. Each frame depicts the Ca2+-binding sites, viz., (a) EF1 and (b) EF2, with the Ca2+-binding residues indicated. The Ca2+ ion in the X-ray structure is shown as a yellowcolored transparent sphere. This figure was drawn using PyMOL (http://www.pymol.org).

stating that the Glu60 residue is not only related to the local coordination patterns of the Ca2+ ions, but also to the Ca2+binding mechanism.37,38 In Ca2+-binding proteins, it was hypothesized that binding of Ca2+ is synchronized with conformational changes based on the central structure between the two Ca2+-binding loops. The structure is named the EFβscaffold, which comprises a small β-sheet.39 Because Glu60 belongs to the EFβ-scaffold, we propose that the nature of the first Ca2+-binding is important not only for gaining the solvation free energy but also for determining the conformational change in Ca2+-binding loops. The overall energy balance between protein structure and desolvation allows fine-tuning of Ca2+-induced conformational changes in Ca2+-binding proteins.

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CONCLUSIONS In this study, we have presented the theoretical results for Ca2+ ion binding by calbindin D9k based on 3D-RISM theory. The ion distribution around several structures of calbindin D9k in a CaCl2 electrolyte solution was evaluated by assessing the distribution and solvation free energy and agreed well with the experimental data. Spatial decomposition analyses were performed for the holo- and apo-states after obtaining the solvation free energy. We identified the residues that contribute primarily to the stabilization of the holo-structure against all apo-structures. The gain in solvation free energy in the holostructure is caused by exposure to solvent. 11502



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Molecular Recognition Study on the Binding of Calcium to Calbindin

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