Probing the Surface Calcium Binding Sites of Melanosomes Using

Aug 26, 2014 - ABSTRACT: Melanosomes have the capacity to bind significant concentrations of calcium, suggesting there are surface binding sites that ...
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Probing the Surface Calcium Binding Sites of Melanosomes Using Molecular Rulers Keely Glass,†,§ Rolando Rengifo,† Fiona Porrka,† and John D. Simon*,†,‡ †

Department of Chemistry, Duke University, Durham, North Carolina 27708, United States Department of Chemistry, Elon University, Elon, North Carolina 27244, United States ‡ Department of Chemistry, University of Virginia, Charlottesville, Virginia 22904, United States §

ABSTRACT: Melanosomes have the capacity to bind significant concentrations of calcium, suggesting there are surface binding sites that enable cations to access the interior of fully pigmented melanosomes. The surface of melanosomes is known to contain significant concentrations of carboxylate groups which likely are the initial biding sites for calcium, but their arrangement on the surface of the melanosome is not known. In various calcium proteins, a bidentate coordination by two carboxylate groups is the most common structure. In this study, we determine the distance between neighboring surface carboxylic acid groups by examining the binding of a series of diamines +H3N(CH2)mNH3+ (m = 1−5) to melanosomes isolated from the ink sacs of Sepia of f icinalis and bovine choroid tissue. Of these amines, ethylenediamine (m = 2) shows optimal bidentate binding, revealing a narrow distribution of distances between neighboring carboxylic acid groups, ∼480 pm, similar to that found in proteins for calcium binding motifs involving two carboxylate groups.

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metals like Ca2+. However, the distribution of the carboxylate groups on the surface and whether the ions bind to a single carboxylate group or to more than one group remain unknown. In this paper, we provide the first insights into the organization of carboxylate groups on the surface of the melanosome. This approach takes advantage of the chelate effect, in which bidentate ligands can form more stable complexes than their monodentate ligand analogues. Compare, for example, the coordination of Cu2+ by ethylenediamine (en) to that of two methylamines (MeNH2). For a given copper concentration where the concentration of methylamine is twice that of ethylenediamine (so that the available “amine” concentration is constant), the concentration of the Cu2+(en) complex is larger than that of the Cu2+(MeNH2)2 complex. As long as the geometry favors dual coordination by the bidentate ligand, the equilibrium binding constant for the bidentate ligand will be larger than that for the monodentate ligands because of the contribution of entropy to the binding constant. Only one en ligand is required to form a complex with Cu2+ (two molecules), while two methylamines must associate to form a complex with Cu2+ (three molecules). Here we used the potential of the chelate effect to determine whether or not the carboxylic acid groups on the surface of

INTRODUCTION Melanin, a major component of melanosomes, has a high affinity for metal ions.1−6 Recent insights into the roles of metal ions in metabolism and awareness of the tight regulations on metal ion concentrations in biological systems have motivated efforts to understand the metal binding affinity, capacity, and molecular details of melanin’s binding sites. Overall, two themes pervade this literature depending on the particular metal ion being examined: the ability of melanin to serve as a reservoir for metal ionsenabling storage, release, and exchangeexemplified by calcium and the ability of melanin to strongly bind and sequester reactive metals that might otherwise induce oxidative stressexemplified by iron.2,7−9 The concentration of binding sites on the melanosome surface is difficult to determine due to the limited understanding of the molecular structure of melanin and its organization within the melanosome. As the concentration of binding sites must be expressed in terms of the amount of melanin (e.g., grams) rather than the actual number of available sites, the binding constants of most metals to melanin remain unknown. In order to quantify and understand the metal binding sites on melanin, new methods must be developed that elucidate the surface structure. To understand how metals are trafficked within the organelle, the chemical structure of the metal binding sites on the melanosome surface must be known. Spectroscopic studies establish that carboxylate groups are the major negatively charged chemical moiety on the surface of the melanosome.10 Therefore, it is reasonable to assume that carboxylic acid groups serve as the initial binding sites for © XXXX American Chemical Society

Special Issue: Spectroscopy of Nano- and Biomaterials Symposium Received: May 31, 2014 Revised: August 24, 2014

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intact S. of f icinalis and bovine choroid melanosomes were arranged at specific distances, or randomly distributed. Specifically, the binding of a series of alkylamines (mono and diamines) to both melanosomes was examined.

5,6-dihydroxyindole-2-carboxylic acid (DHICA) and 5,6dihydroxyindole (DHI).12,14 Previously reported XPS analyses provide insight into the functional groups present on and near the surface of the melanosome; specifically, from highresolution XPS data of the carbon peak, the percentages of carbon present as CHx, CNH2; CO, C2NH; CO; and COO can be determined. For choroid melanosomes and sepia melanin, ∼3 and 4% of the carbon present is present as carboxylate groups, respectively.15,16 These carboxylate groups can be attributed mostly to DHICA.14 The binding of the mono- and diamines to sepia and choroidal melanosomes was examined at pH 5.8. At this pH, the carboxylic acid groups on the surface of the melanosome are deprotonated (pKa ∼ 4.5) and the amine groups are protonated (pKa values are listed in Table 1).17−20 Isothermal titration calorimetry was used to measure the binding isotherms, which were then analyzed to determine the binding equilibrium constants. For monoamines, a single interaction occurs; for diamines, two carboxyl groups can bind as long as the space between the carboxyl groups on the surface is accessible by the diamine. The series of diamines examined, + H3N(CH2)mNH3+ (m = 1−5), probe different distances. The distance between the two nitrogen groups, associated with the lowest energy conformation of the diamine, was determined using AM1 calculations. These values are presented in Table 1. The ITC data for the binding of ethylamine and ethylenediamine to S. off icinalis melanosomes are shown in parts A and B of Figure 2, respectively. To extract the binding constant from the data, we assume an equilibrium model where the melanosome has a single set of equivalent binding sites. In this case, we obtain an excellent fit and a binding constant of 685 for ethylamine and 6.13 × 103 for ethylenediamine. The results of all of the titrations are presented in Table 1. The binding constants of the monoamines (methyl, ethyl, and propyl) reflect the formation of a single amino−carboxyl salt bridge. The equilibrium constants for the three monoamines are similar, suggesting that lengthening the alkyl chain does not alter the binding thermodynamics. The equilibrium constant of methylenediamine is also similar to the monoamines, which indicates that only one of the amino groups of this diamine binds to the surface of the melanosome. The binding constants for the diamines +H3N(CH2)mNH3+ (m = 2−5) show a similar trend for both melanosomes studied. Each of these diamines exhibits a greater binding constant than the monoamines, which suggests that, unlike methylenediamine, both amino groups of the diamines interact with the melanosome surface to some extent. For both S. of ficinalis and bovine choroid melanosomes, ethylenediamine exhibits the greatest binding constant. Distortion of the diamine molecule from its equilibrium configuration to enable bidentate binding of the surface carboxyl groups would result in a decrease in the binding constant. Thus, the increased stability observed for the binding of ethylenediamine means that the distance between the two amino groups in the equilibrated structure is best matched to the spacing between carboxylic acid groups on the melanosome surface. This series of molecular rulers therefore reveals that the carboxylate groups are not randomly distributed along the surface of the melanosome but rather that there is a substantial population of carboxylate groups separated by the distance accessed by ethylenediamine. The amines studied can also interact with the MES buffer used to establish the pH of the solutions. MES contains negatively charged groups (in this case sulfonic acid groups),

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EXPERIMENTAL METHODS To acquire scanning electron microscopy (SEM) images, 2 μL aliquots of 1 mg/mL suspensions of S. off icinalis and bovine choroid melanosomes in ultrapure deionized water were deposited on carbon tape and dried in the dark under N2 flow. Images of melanosomes isolated from S. off icinalis were captured on an FEI XL30 FEG-SEM operated at 7 kV in ultrahigh-resolution mode with a spot size of 3 and a working distance of 6.4 mm. Images of melanosomes isolated from bovine choroid tissue were captured on an FEI Quanta 650 FEG-SEM operated at 5 kV in ultra-high-resolution mode with a spot size of 3 and a working distance of 9.7 mm. To increase resolution, S. off icinalis melanosomes were coated with a 10 nm layer of Au/Pd with a Hummer V sputter coater and choroid melanosomes were coated with a 30 nm layer of Au/Pd with a GATAN etching and coating system. Isolation of melanosomes from S. off icinalis ink sacs and bovine choroid tissue was conducted as previously reported.11−13 The melanosomes were treated with ethylenediaminetetraacetic acid (EDTA) to remove the metal ions bound to the surface moieties. The amines were purchased as hydrochloric acid salts from Sigma-Aldrich. Amine concentrations used in experiments with S. off icinalis and bovine choroid melanosomes were 6 and 9 mM, respectively. The MES buffer (2-(N-morpholino)-ethanesulfonic acid) was prepared from low moisture content, ≥99% (titration) MES from Sigma-Aldrich and the pH was adjusted with 1 M NaOH to 5.8 using a pH meter. Equilibrium binding constants were determined with isothermal titration calorimetry (VP-ITC MicroCalorimeter, Microcal). As the molecular weight and thus molar concentration of melanin is not known, the equilibrium concentration is expressed as mg of melanin/mL rather than moles. As long as the concentrations of melanin and amines are held constant for a particular study (e.g., using aliquots of the same choroid melanosome solution for all titrations and injecting the same concentrations for all amines studied), the relative values of the binding constant are reflective of thermodynamic changes.

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RESULTS AND DISCUSSION Figure 1 shows a SEM image of the two samples examined. Sepia melanosomes are spherical with an average diameter of ∼150 nm.11 Choroid melanomes are larger and ellipsoidal in shape, with a long axis of ∼860 nm and an aspect ratio of ∼0.14.12 Detailed chemical analyses of these samples establish that they both contain only eumelanin which is composed of

Figure 1. SEM images of Sepia of f icinalis (A) and bovine choroid melanosomes (B). B

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Table 1. Binding Constants, pKa Values, and AMI Calculations of a Series of Amines to S. off icinalis (Sepia) and Ocular Bovine Choroid (Choroid) Melanosomes amine

pKa1

pKa2

ra (pm)

Ksepiab

methylenediamine ethylenediamine 1,3-diaminopropane 1,4-diaminobutane 1,5-diaminopentane 1-methylamine 1-ethylamine 1-propylamine

10.44 7.56 9.03 9.71 10.05 10.63 10.70 10.65

10.44 10.71 10.94 11.15 10.93

225 297 362 476 444

243 6.13 × 1003 4.76 × 1003 1.91 × 1003 956 69.6 685 142

Kchoroid 176 5.92 1.81 1.83 1.96

× × × ×

1003 1003 1003 1003

a The distance, r, reported here is the distance between the centers of the two N atoms for the lowest energy configuration of the amine. bThe values for the diamines listed are the average of three determinations.

Figure 2. Binding isotherms of ethylamine (A) and ethylenediamine (B) measured with an isothermal titration calorimeter.

association constant for Ca2+ binding to S. of f icinalis melanosomes is 3.3 (±0.2) × 103 M−1, which is comparable to intracellular calcium binding proteins that serve to buffer calcium concentrations.25 IR spectroscopy of S. off icinalis bound to Ca2+ reveals that the COOH stretching vibration at 1710 cm−1 decreases with increasing metal concentration, indicating the loss of a proton in the presence of metal.10 The capacity of the melanosome for Ca2+ is ∼1.5−1.6 mmol/g or roughly one Ca2+ for every four “monomers” in the melanin pigment.26 Scanning electron microscopy (SEM) and atomic force microscopy (AFM) studies confirm that the binding does not create a coating of “Ca2+” material on the surface.27 The ions are transported and permeate the entire melanosome. The saturation level and associated spectral changes that occur with binding suggest similar environments for the bound Ca2+ ions. This is consistent with the idea that melanosomes possess channels and that calcium binds to the surface of these channels and travels through them into the melanosome bulk. As a divalent cation, coordination with two carboxylic acid groups is necessary to neutralize the charge on calcium.

which can form ionic bonds with the ammonium groups. Even in a fully buffered system where a solution of the amine is titrated into pure buffer, a heat greater than the dilution heat is observed, reflecting the changes in the concentrations of free amine and amine−MES associations as equilibrium is reestablished. This contribution to the heat does not affect the trend observed for the binding of the amines to the melanin samples but would affect the magnitude of the equilibrium constant determined from the ITC analysis. This means that the equilibrium constants in Table 1 are underestimates of the true equilibrium constants and differ from the actual equilibrium constants by a multiplicative factor equal to the equilibrium constant associated with the amine−MES interaction.21 The findings present in Table 1 have a significant impact on our understanding of how Ca2+ binds to melanosomes. This is an important topic, as the regulation of calcium in melanocytes may affect numerous biological pathways.2,22,23 In the cell, redox chemistry is protected by calcium and the regulation of Ltyrosine, a substrate required in melanogenesis.24 The C

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Studies of the crystal structures of a large number of calcium−carboxylate complexes reveal three different binding motifs: the monodentate interaction of the metal with the oxygen of one carboxylate group, the bidentate coordination of the metal to the oxygen of two different carboxylate groups, and the bidentate interaction of the metal with one oxygen of a carboxylate group and an oxygen or nitrogen bound to the αcarbon of the carboxylate group.28−31 The former bidentate interaction is the most common structure, and the latter likely does not apply in the case of melanin binding, as the aromatic structure of the pigment precludes the presence of appropriate binding groups on the carbons α to the carboxylate groups. For monodentate complexes, the Ca2+−O distance is ∼238 pm (it is slightly larger, 242 and 253 pm, for the two bidentate complexes, respectively). If the surface binding of Ca2+ involves coordination of two carboxylic acid groups, the above data suggests that optimal binding would be achieved if the distance between the coordinating oxygen atoms of the two carboxyl groups was ∼480 pm. The distances reported in Table 1 are a measure of center−center distances between the nitrogen atoms for the lowest energy conformations of the diamines. The actual binding distance must take into account the N−H bond distance and the hydrogen bond length. To estimate these, we use the results for the crystal structure of ammonium acetate, where the N−H and hydrogen bond have lengths of 90 and 280 pm, respectively.32 The lowest energy structure of the diamines does not result in linear structures with the amines pointing away from one another; in the case of the ethylenediamine, the normal to the NH3 planes is at an ∼30° angle, so the actual distance between the two coordinated oxygen atoms is 180 pm longer than the N−N distance calculated, or ∼480 pm, which is that predicted for using the coordination sphere data of Ca2+. In the case of the longer diamines, the normals to NH3 are at a larger angle (∼130, 45, 90° for n = 3, 4, and 5) and a distance >180 pm needs to be added to the N−N distance calculated. The binding and transport of calcium into the melanosome interior involves an interaction with the surface of the melanosome. On the basis of published data detailing the interaction of calcium with carboxylate residues and the observations herein, it is clear that the carboxylic acid groups are ordered on the surface of the melanosome to bind metals such as Ca2+. The distance observed between carboxylic acid groups corresponds well to previous studies on calcium and emphasizes the importance of the structure in understanding the function of melanosomes in nature.

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AUTHOR INFORMATION

Corresponding Author

*Phone: 434-982-2362. Fax: 434-924-1497. E-mail: john. [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We thank Mostafa El-Sayed for inspiring us to think of using molecules as rulers. We thank Duke University and University of Virginia for support of this work. We also thank Richard White and the NMCF at University of Virginia for the operation and use of facilities’ instrumentation. D

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