Article Cite This: Inorg. Chem. XXXX, XXX, XXX−XXX
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How Pb2+ Binds and Modulates Properties of Ca2+-Signaling Proteins Todor Dudev,*,† Cédric Grauffel,‡ and Carmay Lim*,‡,§ †
Faculty of Chemistry and Pharmacy, Sofia University, Sofia 1164, Bulgaria Institute of Biomedical Sciences, Academia Sinica, Taipei 11529, Taiwan § Department of Chemistry, National Tsing Hua University, Hsinchu 300, Taiwan ‡
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S Supporting Information *
ABSTRACT: Abiogenic lead (Pb2+), present in the environment in elevated levels due to human activities, has detrimental effects on human health. Metal-binding sites in proteins have been identified as primary targets for lead substitution resulting in malfunction of the host protein. Although Pb2+ is known to be a potent competitor of Ca2+ in protein binding sites, why/how Pb2+ can compete with Ca2+ in proteins remains unclear, raising multiple outstanding questions, including the following: (1) What are the physicochemical factors governing the competition between Pb2+ and Ca2+? (2) Which Ca2+-binding sites in terms of the structure, composition, overall charge, flexibility, and solvent exposure are the most likely targets for Pb2+ attack? Using density functional theory combined with polarizable continuum model calculations, we address these questions by studying the thermodynamic outcome of the competition between Pb2+ and Ca2+ in various model Ca2+-binding sites, including those modeling voltage-gated calcium channel selectivity filters and EF-hand and non-EF-hand Ca2+-binding sites. The results, which are in good agreement with experiment, reveal that the metal site’s flexibility and number of amino acid ligands dictate the outcome of the competition between Pb2+ and Ca2+: If the Ca2+-binding site is relatively rigid and crowded with protein ligands, then Pb2+, upon binding, preserves the native metal-binding site geometry and at low concentrations, can act as an activator of the host protein. If the Ca2+-binding site is flexible and consists of only a few protein ligands, then Pb2+ can displace Ca2+ and deform the native metalbinding site geometry, resulting in protein malfunction.
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INTRODUCTION Over the course of cell evolution, about 24 (biogenic) metal species have been selected and assigned biological functions based on their intrinsic physicochemical properties and bioavailability.1 The most frequently found biogenic metal ions are Na+, K+, Mg2+,Ca2+, Zn2+, Mn2+, Fe2+/3+, Co2+/3+, Ni2+, and Cu+/2+, which play vital roles in a multitude of essential tasks such as protein structure stabilization, enzyme catalysis, blood coagulation, signal transduction, muscle contraction, hormone secretion, taste and pain sensation, respiration, and photosynthesis.1−5 Other abiogenic/xenobiotic metal species, e.g., Hg2+, Pb2+, Al3+ or Tl+/Tl3+, upon entering organisms lacking efficient defensive mechanisms against such intruders, can adversely affect the cellular processes by competing with the native cations in various proteins. This is the essence of the dominating perception of the heavy-metal poisoning mechanism postulating that the xenobiotic metal cation disrupts the structure of the protein active site(s) upon binding, thus compromising the normal functions of the host metalloprotein.6−12 Lead (Pb2+), a dication in group IVA of the periodic table with unknown biological function in higher organisms, is commonly found in water and soil at concentrations ∼1000fold higher than its natural levels as a result of using leaded paint, leaded fuel, and lead-containing water pipes.12−14 It has © XXXX American Chemical Society
detrimental effects on human health, as manifested by severe neurological, cardiovascular, reproductive, renal, endocrine, hematological, and/or immune dysfunctions.12 This is largely because lead is a potent neurotoxin that interferes with signaling cascades in the brain causing cognitive and psychiatric disorders.15 The wide variety of lead-caused pathology suggests that multiple protein targets for lead’s actions in vivo exist. Metal-binding sites in proteins that are primary targets for lead attack have been identified: These are zinc centers comprising multiple sulfhydryl/thiolate ligands (Cys0/Cys− residues) or Ca2+-binding sites with oxygencontaining ligands such as Asp−/Glu− and Asn/Gln side chains and backbone carbonyls.13 Such oxygen-rich Ca2+-sites are present in proteins involved predominantly in cell signaling, e.g., voltage-gated calcium channels in neurons12,16−20 and EFhand proteins.11,12,21−24 Voltage-gated calcium channels are involved in variety of essential tasks such as cell signaling, muscle contraction, hormone secretion, gene expression regulation, and neurotransmitter release.25 They passively transport Ca2+ ions from the extracellular to the intracellular space along the concentration gradient and are highly Ca2+selective due largely to a tetrameric EEDD and EEEE Received: September 7, 2018
A
DOI: 10.1021/acs.inorgchem.8b02548 Inorg. Chem. XXXX, XXX, XXX−XXX
Article
Inorganic Chemistry selectivity filter comprising Asp− (D) or Glu− (E) carboxylates.26 EF-hand proteins belong to a large family of cytoplasmic Ca2+-binding proteins involved in signal transduction, protein metabolism, and cell dynamics and differentiation. They are so-called as they contain a well-defined helix−loop−helix EF-hand motif whose metal center is highly selective for the cognate Ca2+ over other biogenic metal species in the surrounding fluids such as Mg2+, K+, and Na+.27 Although Pb2+ is known to be a potent competitor of Ca2+ in protein binding sites, why/how Pb2+ can compete with Ca2+ in proteins is not well understood. For the same metal coordination number (CN), Pb2+ has a larger ionic radius than Ca2+, e.g., 1.19 versus 1.00 Å for a CN of 6 and 1.23 versus 1.06 Å for a CN of 7,28 and longer Pb2+−ligand distances.29 The coordination geometries of protein-bound Pb2+ and Ca2+ also differ: Pb2+ can adopt either a holodirected nearly spherical geometry where the ligating atoms are uniformly distributed around Pb2+ or a hemidirected geometry where the ligating atoms are clustered in one hemisphere while its 6s2 lone pair occupies the other hemisphere. Due to relativistic effects, the 6s2 electron pair is contracted and rarely participates in bond formation.14,30 Its stereochemical activity depends largely on the number of ligating entities, i.e., the metal CN: A less crowded sphere favors hemidirected Pb2+ coordination geometry with a stereochemically active 6s2 lone pair, whereas larger coordination numbers are compatible with holodirected spherical geometry where strong steric and electrostatic interactions among the ligands render the lone pair stereochemically inactive.30,31 In contrast to Pb2+, Ca2+ seldom adopts hemidirected geometry except in a few non-EFhand Ca2+-binding sites.29 Since Pb2+ does not mimic Ca2+, this raises the following important fundamental questions: (1) Compared to Ca2+, Pb2+ is more electronegative32 but is larger and forms longer bonds (which affects the amount of ligand → metal charge transfer), so which dication would be a better electron acceptor? (2) What are the physicochemical factors governing the selectivity for Pb2+ over Ca2+? (3) What type of Ca2+-binding sites in terms of the structure, composition, overall charge, flexibility, and solvent exposure are the most likely targets for Pb2+ attack? Here, we endeavor to shed light on these questions by studying the thermodynamic outcome of the competition between Pb2+ and Ca2+ in various model Ca2+-binding sites. Density functional theory (DFT) calculations in combination with polarizable continuum model (PCM) computations were employed (see the “Methods” section). The competition between Pb2+ and Ca2+ can be expressed in terms of the free energy, ΔGε, for replacing the native Ca2+ cation bound to the protein by its “alien” rival, Pb2+:
composition, charge state, and solvent exposure of the metal site. Trends in the free energies computed using this approach have been found to be consistent with experimental observations in previous studies.33−39 Next, we computed the free energies for eq 1 in Ca2+-binding sites modeling voltagegated calcium channel selectivity filters, which are known to mediate Pb2+-entry into the brain.11 We also computed the Ca2+ → Pb2+ exchange free energies in EF-hand Ca2+-sites in calmodulin, a protypical EF-hand Ca2+-binding protein as well as in non-EF-hand Ca2+-sites in signaling protein S since their Pb2+- and/or Ca2+-bound X-ray structures are available. The results, which are in good agreement with experiment, reveal that the metal site’s flexibility and number of amino acid (aa) ligands govern the outcome of the competition between Pb2+ and Ca2+. They also reveal how these two factors modulate the properties of the lead-substituted metal center.
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[Pb2 +‐aq] + [Ca 2 +‐protein] → [Pb2 +‐protein] + [Ca 2 +‐aq]
METHODS
Models Used. The side chains of Asp−, Glu−, Asn, and Ser, and backbone peptide group were modeled as propionate (CH3CH2COO−), butyrate (CH3CH2CH2COO−), propionamide (CH3CH2CONH2), ethanol (CH3CH2OH) and N-methylacetamide (CH3CONHCH3), respectively. Mononuclear metal-binding sites were considered, whereas polynuclear binding sites will be treated in separate study. In proteins, Ca2+ is often heptacoordinated to aa ligands (denoted as L),40 hence its protein binding sites were modeled as [Ca·(H2O)7−m·Lm] where m = 1−5. The geometryoptimized calcium complexes were used as starting structures for optimizing Pb2+ complexes by replacing Ca2+ with Pb2+. In aqueous solution, the number of first-shell water molecules varies between 6−8 in Ca2+ aqua complexes41 and between 6−9 in Pb2+ hydrates42 with 6 being the most probable number for both dications (see Figures S1 and S2). Accordingly, hexacoordinated aqua complexes were modeled for Ca2+ and Pb2+. Models of EF-hand and non-EF-hand Ca2+-binding sites were derived from the high-resolution X-ray structures of calmodulin (Protein Data Bank (PDB) entry 1EXR) and signaling protein S (PDB entry 1NPS).43 Models of EEDD and EEEE selectivity filters of voltage-gated calcium channels were created following previous work.44 All the structures were built using GaussView version 3.09.45,46 Choice of the Protocol for Geometry Optimization. In proteins, Ca2+ binds mainly to O atoms from backbone or Asn/Gln carbonyl groups, Asp/Glu carboxylates, Ser/Thr hydroxyl groups, and water. Hence, high-resolution structures of Ca2+ and Pb2+ complexes from the Cambridge Structural Database (CSD)47 were chosen if they contained protein-like ligands with neutral O and negatively charged Oneg ligating atoms. The reference structures selected (Table S1) cover CNs ranging from 6 to 9 for Ca2+ and 5 to 8 for Pb2+, whereas the net charge varies between −2 and +2. They include both hemidirected, e.g., CSD entry GICFAB, and holodirected, e.g., CSD entry OSAPEE, geometries for Pb2+ and at least one polynuclear complex for each dication. These reference structures were used to calibrate the geometry optimization method (see below). Many combinations of density functionals and basis sets were assessed for their abilities to reproduce these CSD structures. The 23 functionals tested include the widely used B3-LYP48 (with and without addition of Grimme’s empirical dispersion),49 B-LYP,50 BB1K,51 B97−2,52−54 BMK,55 PBE0,56 BP86,50,57 wB97XD,58 B3PW91,48 MPW1K,59 TPSS,60 S-VWN,61,62 and ten Minnesota functionals (M05,63 M06,64 M06-L,65 M06-2X,64 M11,66 M11-L,67 MN12-L,68 N12,69 N12-SX,69 and MN12-SX).68 Each of these 23 functionals was combined with the 6-31+G* basis set for all atoms except Pb2+, which employed scalar relativistic pseudopotential, LanL2DZ70 or SDD,71 whose reliability in describing lead hydrates has been established by full-relativistic calculations.72 For each functional/6-31+G*(SDD/LanL2DZ) combination, each structure was optimized in a low-dielectric environment (ε = 10) to compare with the X-ray geometries. For each fully optimized structure, we
(1)
In eq 1, [Pb2+/Ca2+-protein] and [Pb2+/Ca2+-aq] represent the metal ion bound to protein ligands inside the metal-binding cavity and unbound in aqueous solution, respectively. Buried and solvent-accessible metal-binding sites are characterized by an effective dielectric constant, ε, of ∼4 and ∼30, respectively, whereas bulk aqueous solvent is represented by ε = 78. A positive ΔGε implies a Ca2+-selective site, whereas a negative value implies a Pb2+-selective one. To extract the key factors underlying the Pb2+ versus Ca2+ competition, we examined how the free energy for eq 1 changed with varying structure, B
DOI: 10.1021/acs.inorgchem.8b02548 Inorg. Chem. XXXX, XXX, XXX−XXX
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Inorganic Chemistry
Figure 1. (A) Deviation of the M−O distances computed using MPW1K/6-31+G(d)(SDD) at ε = 10 (top) from the respective X-ray values (yaxis) for each of the CSD structures in Table S1. (B). Calculated cation exchange free energies in aqueous solution (ε = 78.35) and optimized structures of Ca2+ and Pb2+ complexes with EDTA, BCPE, and TPEN. modeled herein. The overall geometry of each lead complex was wellpreserved: The L−M−L angles are in agreement with those observed in the X-ray structures (Table S1) with an average root-mean-squaredeviation (RMSD) of 0.36 Å for the heavy atoms. The geometry of a metal complex is relatively insensitive to the environment:73 When the CSD complexes were optimized with ε = 30 or 78, the δ values in Figure S3 remain comparable to those displayed in Figure 1A. Choice of the Protocol for Energy Calculations. Since the MPW1K/6-31+G(d)//SDD method best reproduced the geometries in the data set of CSD structures, it was used to optimize the geometries of all metal complexes in a polarizable continuum model.
computed the metal−ligand (M−L) distances and averaged equivalent M−L distances to yield a mean value. We also measured at least one L−M−L angle in each Pb2+-containing structure; these angles are shown in Table S1. Among the different methods tested, the MPW1K/6-31+G*(SDD) method best reproduced the M−L distances in all the CSD structures with an average deviation from the respective X-ray distances of 0.05 Å and a maximal deviation of 0.17 Å. Figure 1A shows the deviation δ of the MPW1K/6-31+G*(SDD) M−L distances from the respective X-ray values (y-axis) for each of the structures tested (x-axis). The metal complexes bound only to O/Oneg atoms are related to those C
DOI: 10.1021/acs.inorgchem.8b02548 Inorg. Chem. XXXX, XXX, XXX−XXX
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Inorganic Chemistry
to Ca2+, regardless of the charge scheme (NBO or Hirshfeld) employed or type of ligand in Table 1. Thus, Pb2+ could be a strong competitor of Ca2+ in proteins. Before determining which Ca2+-binding sites in proteins are vulnerable to Pb2+ attack, we first assessed how the outcome of the Pb2+ vs Ca2+ competition depends on the number, type/charge of the metal ligands, and the local environment, modeled by an effective dielectric constant, ε. Metal Complexes Containing Neutral Ligands. First, we examined how replacing Ca2+ with Pb2+ affects the structures of the metal complexes containing varying numbers of neutral amide ligands. We constructed heptacoordinated Ca2+ complexes containing 2, 3, 4, and 5 N-methylacetamide ligands (modeling backbone carbonyl groups in proteins) complemented by the respective number of water molecules and optimized the geometries in low-dielectric (ε = 4) and higher-dielectric (ε = 29) media. The resulting geometries optimized in ε = 4 are similar to those optimized in ε = 29, consistent with previous findings that the environment does not significantly perturb the metal coordination geometry.73 The Ca2+ ion in each fully optimized geometry was then replaced by Pb2+ and reoptimized to yield the respective lead complexes. Comparison of the optimized structures of Ca2+ and Pb2+ complexes shows varying degree of conformational changes depending on the number of bound ligands. In lighter complexes with ≤4 amide ligands (Figure 2A−C), significant geometrical rearrangements occurred upon Ca2+ → Pb2+ substitution: The amide ligands which were evenly distributed around Ca2+ isomerized spontaneously to a hemidirected arrangement where they banded together in one hemisphere, liberating space for the 6s2 lone pair in the other hemisphere. Furthermore, a first-shell water molecule was relegated to the metal’s second shell, thus decreasing the metal CN to 6. The heaviest complex with five amide ligands, however, retained the holodirected geometry of the Ca2+ complex with the ligands distributed around the metal center as well as calcium’s CN of 7 (Figure 2D). Our findings are consistent with statistical analyses of Pb2+ and Ca2+ complex structures in the CSD showing that Pb2+ complexes with crowded coordination spheres favor a spherical holodirected geometry, whereas those with smaller and/or fewer ligands generally adopt a hemidirected geometry.78 Because Pb2+ is a better electron acceptor than Ca2+, the net amount of charge transferred from the ligands to Pb2+ is greater than that to Ca2+ (Table 2), thus enhancing the Pb2+− ligand interactions. Furthermore, the charge-accepting ability of Ca2+ saturates faster than that of Pb2+: After charge transfer from the first amide, charge transfer from successive replacement of a water with an amide is nearly zero for Ca2+ but steadily decreases from 0.026 e to 0.012 e to 0.003 e in the Pb2+ complexes (Table 2). These differences enable Pb2+ to displace Ca2+ heptacoordinated to ≤4 amide ligands in buried sites (negative ΔG4 in Figure 2). However, despite the steady increase in the net charge transferred to Pb2+ with an increasing number of bound amides, the Ca2+ → Pb2+ substitution in the penta-amide complex (Figure 2D) is less favorable than that in the lighter complexes (Figure 2A−C). This may reflect lead’s preference for hemidirected coordination that allows the 6s2 electron lone pair to reside in a hemisphere rather than holodirected coordination where the lone pair is squeezed between the heavy amide ligands.29,78 Increasing solvent exposure of binding sites lined by >2 neutral
Note that the SDD scalar relativistic pseudopotential, which can reproduce several experimental observables for a series of heavy-metal systems,71 takes into account the relativistic effects on Pb2+. Stability constants at 298 K for both Ca2+ and Pb2+ in complex with EDTA, BCPE, and TPEN chelating agents,74 which contain 4, 2, and 0 carboxylate groups, respectively (see Figure 1B), were used to select the best protocol for the free energy calculations. The initial structure of the Pb2+/EDTA complex was based on the X-ray structure (CSD entry LORLOT), whereas that of Pb2+ in complex with BCPE or TPEN was derived from a homologous X-ray structure (CSD entry HUSZOL). These initial structures were optimized in water (ε = 78.35) using the MPW1K/6-31+G(d)//SDD method. The mean deviations of the Pb2+−L distances in the fully optimized EDTA, BCPE, and TPEN complexes from the respective X-ray values are +0.01, −0.05, and −0.02, respectively. Pb2+ was then replaced by Ca2+ and reoptimized to yield the respective calcium complexes. On the basis of these fully optimized metal complexes, the free energies, ΔGε, at T = 298 K for the reactions displayed in Figure 1B were estimated according to ΔGε ∼ ΔE ε elec + ΔE ε therm − T ΔS ε
(2)
where ΔEεelec, ΔEεtherm, and ΔSε are the changes in the electronic energy, thermal energy, and entropy, respectively. To compute Eslnelec, single-point energy calculations were performed using the SMD solvation model75 with the same set of 23 functionals listed above combined with SDD or LanL2DZ for Pb2+ and 6-31+G(2d,2p), 631+G(3d,p), 6-311++G(2d,2p), or 6-311++G(3d,p) for the other atoms. The thermal energy and entropy were derived from standard statistical thermodynamics formulas using the MPW1K/6-31+G(d) vibrational frequencies scaled by an empirical factor of 0.9515.76 Among all combinations tested, B3LYP/6-31+G(2d,2p)(SDD) with the addition of Grimme’s empirical dispersion49 led to the best reproduction of experimental free energies with deviations of 0.17, 0.16, and 0.11 kcal/mol for the EDTA, BCPE, and TPEN complexes, respectively (see Figure 1B). Therefore, it was used to compute the Ca2+ → Pb2+ free energies for eq 1 in low-dielectric solvent (diethyl ether, ε = 4.24) to mimic buried sites as well as high-dielectric solvent (propanonitrile, ε = 29.3) for more solvent-exposed sites.
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RESULTS To determine which dication is the better electron acceptor, we computed the charge transferred from water and various aa ligands to the metal ion in Ca2+ and Pb2+ complexes. In agreement with the greater electronegativity32 of Pb2+ (1.6) as compared to that of Ca2+ (1.0), Pb2+ is a better electron acceptor than Ca2+ (Table 1), as it is more polarizable and can therefore accept more charge from a given ligand. The charge transferred to Pb2+ is significantly greater than that transferred Table 1. Amount of Charge (in Electrons) Transferred from the Ligands to the Metal Ion in Ca2+ and Pb2+ Complexesa metal complex
charge transfer to Ca2+ (e)
charge transfer to Pb2+ (e)
[M·H2O]2+ [M·CH3CONHCH3]2+ [M·CH3CH2OH]2+ [M·CH3CH2COO−]+ [M·CH3CH2S−]+
0.02 0.03 0.04 0.12 0.33
0.07 0.17 0.16 0.36 0.68
a
The amount of charge transferred from the ligands to the dication is equal to 2 minus the dication’s charge in the metal complex, which was computed using the NBO77 scheme at the B3LYP/6-31+G(2d,2p)(SDD) level based on MPW1K/6-31+G*(SDD) optimized geometries. Although not shown, we also computed Hirshfeld charges, which like the NBO charges, yield greater charge transfer from the ligand to Pb2+ than to Ca2+. D
DOI: 10.1021/acs.inorgchem.8b02548 Inorg. Chem. XXXX, XXX, XXX−XXX
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Figure 2. Calculated free energies and optimized structures of Ca2+ and Pb2+ complexes with (A) 2, (B) 3, (C) 4, and (D) 5 N-methylacetamide ligands. ΔG4 and ΔG29 refer to cation exchange free energy in an environment characterized by an effective dielectric constant of 4 and 29, respectively.
exceed four.41 In all the fully optimized metal complexes, the carboxylates are monodentately bound to the dication, whereas their metal-free oxygen atoms form hydrogen-bonding interactions with neighboring water molecules. As found in metal complexes containing ≤4 heavy ligands in Figure 2, Pb2+ adopted hemidirected coordination geometry in all the carboxylate complexes. Compared to neutral amide ligands, anionic ligands would be expected to enhance the Pb2+/Ca2+ selectivity due to lead’s ability to accept more charge from the ligands than Ca2+ (Table 1). Indeed, the carboxylate complexes exhibited stronger selectivity for Pb2+ over Ca2+ than the amide complexes: The metal-exchange free energies in Figure 3 are
amide ligands tends to slightly enhance the selectivity for Pb2+ over Ca2+ (the ΔG29 values in Figure 2B−D are more negative than the respective ΔG4 numbers). This is likely due to the solvation free energy gain of outgoing Ca2+ whose hydration free energy (−360 kcal/mol) is more favorable than that of Pb2+ (−341 kcal/mol). Metal Complexes Containing Anionic Ligands. Next, we optimized metal complexes comprising 1−4 propionate ligands (modeling the Asp− side chain) and evaluated the Ca2+ → Pb2+ free energies (Figure 3). Pentacarboxylate metal complexes were not modeled since theoretical calculations and statistical analyses of PDB structures show that the maximum number of carboxylates bound to a dication is unlikely to E
DOI: 10.1021/acs.inorgchem.8b02548 Inorg. Chem. XXXX, XXX, XXX−XXX
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Inorganic Chemistry
more negative than those in Figure 2 by 4−8 kcal/mol. The Ca2+ complexes containing 1−3 carboxylates (Figure 3A−C) exhibit similar Ca2+ → Pb2+ free energies (ΔG4 = −12 kcal/ mol). However, the tetracarboxylate Ca2+ complex (Figure 3D) has a less favorable metal-exchange free energy (−6 kcal/ mol) even though it adopted hemidirected coordination geometry. This is probably because after substantial charge transfer from three carboxylates to the cation, Pb2+, rather than accepting charge, back-donates charge to the ligands (Table 2); thus, the affinity of the tetracarboxylate site for Pb2+ is attenuated. Increasing solvent exposure of the binding site did not enhance the Pb2+/Ca2+ selectivity likely due to the poorer solvation of the bulkier Pb2+ complexes compared to the Ca2+ counterparts, which offsets the solvation free energy gain of the displaced Ca2+ relative to the desolvation penalty of Pb2+. Calcium Channel Selectivity Filters. Vertebrate voltagegated Ca2+ channels possess monolayered tetrameric selectivity filters lined with Asp−/Glu− residues.79−82 Following guide-
Table 2. Charge Transfer and Its Increment (in Parentheses) from the Ligands to the Metal in Ca2+ and Pb2+ Complexesa metal complex
charge transfer to Ca2+ (e)
charge transfer to Pb2+ (e)
[M·(H2O)5·(CH3CONHCH3)2]2+ [M·(H2O)4·(CH3CONHCH3)3]2+ [M·(H2O)3·(CH3CONHCH3)4]2+ [M·(H2O)2·(CH3CONHCH3)5]2+ [M·(H2O)6·CH3CH2COO]+ [M·(H2O)5·(CH3CH2COO)2]0 [M·(H2O)4·(CH3CH2COO)3]− [M·(H2O)3·(CH3CH2COO)4]2−
0.153 0.153 (0.000) 0.154 (0.001) 0.154 (0.000) 0.155 0.166 (0.011) 0.171 (0.005) 0.172 (0.001)
0.358 0.384 (0.026) 0.396 (0.012) 0.399 (0.003) 0.395 0.417 (0.022) 0.453 (0.036) 0.436 (−0.017)
a
The amount of charge transfer was computed using the NBO77 scheme at the B3LYP/6-31+G(2d,2p)(SDD) level based on MPW1K/6-31+G*(SDD) optimized geometries. The number in parentheses is the increment in charge transfer upon successive replacement of a water molecule with an amide or carboxylate.
Figure 3. Calculated free energies and optimized structures of Ca2+ and Pb2+ complexes with (A) 1, (B) 2, (C) 3, and (D) 4 propionate ligands. ΔG4 and ΔG29 refer to cation exchange free energy in an environment characterized by ε = 4 and 29, respectively. F
DOI: 10.1021/acs.inorgchem.8b02548 Inorg. Chem. XXXX, XXX, XXX−XXX
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Figure 4. Calculated free energies and optimized structures of Ca2+ and Pb2+ bound to model tetrameric selectivity filters comprising (A) EEDD and (B) EEEE loci. ΔG4 and ΔG29 refer to cation exchange free energy in an environment characterized by ε = 4 and 29, respectively.
Figure 5. Calculated free energies and optimized structures of Ca2+ and Pb2+ bound to model (A) EF-II/EF-III and (B) EF-I/EF-IV binding sites.
lines from our previous work,44 we optimized metal-bound tetrameric selectivity filters comprising EEDD (Figure 4A) and EEEE residues (Figure 4B) with D and E modeled by CH3CH2COO− and CH3CH2CH2COO−, respectively; we refer the reader to previous work for a justification of the models used.35,36 Unlike Ca2+, which binds in the plane formed by the four monodentately bound carboxylate oxygen atoms, the larger Pb2+ binds out of this plane, coordinating to one of the carboxylates bidentately, increasing its CN to 5. In solventinaccessible pores where electrostatic interactions predominate, both EEDD and EEEE selectivity filters have higher affinity for the stronger charge acceptor, Pb2+ (Figure 4, negative ΔG4). Solvent entry into the selectivity filter screens the favorable metal−COO− electrostatic interactions and
results in poorer solvation of the bulkier penta-coordinated Pb2+ complexes compared with the tetra-coordinated Ca2+ counterparts, thus reducing Pb2+/Ca2+ selectivity in the solvent-exposed pore (Figure 4, ΔG29 is less negative than ΔG4). Hence, “alien” Pb2+ is predicted to irreversibly bind and occlude EEDD and EEEE selectivity filter pores. EF-Hand Binding Sites. The classical EF-hand motif comprises a 12-residue Ca2+-binding loop flanked by two helices forming a conserved helix−loop−helix structure.83 The Asp−/Glu− side chains and Asn/Gln/backbone carbonyls from the loop coordinate Ca2+ in pentagonal bipyramidal geometry.84−86 We modeled the two types of classical EF-hand binding sites in calmodulin, a key protein involved in cell signaling:29,87 EF-II and EF-III sites comprising 2 Asp−, 1 G
DOI: 10.1021/acs.inorgchem.8b02548 Inorg. Chem. XXXX, XXX, XXX−XXX
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Figure 6. Calculated free energies and optimized structures of Ca2+ and Pb2+ bound to model (A) site 1 and (B) site 2 of protein S.
Glu−, and 1 Asn side chains and a backbone carbonyl group (Figure 5A) and EF-I and EF-IV sites comprising 3 Asp− and 1 Glu− side chains and a backbone carbonyl group (Figure 5B). A water molecule complements the ligation sphere in the two types of EF-hand sites, which are all solvent-exposed. The calculations could reproduce the Ca2+−O bond distances in the 1.0 Å X-ray structure of calmodulin (PDB entry 1EXR): The mean experimental (2.40 Å) and computed (2.46 Å) Ca2+−O distances in the EF-II/III binding sites exhibit a RMSD of 0.06 Å, whereas those in the EF-I/IV binding sites, 2.39 and 2.49 Å, exhibit a RMSD of 0.10 Å. Lead, upon replacing Ca2+, preserves the overall shape of these congested binding pockets and the native holodirected geometry (Figure 5). Consistent with the trends found for the amide and carboxylate-containing metal complexes in Figures 2 and 3, the EF-hand binding sites exhibit higher affinity toward the “alien” cation relative to the native Ca2+ (Figure 5, negative ΔG4/ ΔG29). Also consistent with the trend found in Figure 3, it is thermodynamically more favorable to replace Ca2+ with Pb2+ in the three-carboxylate EF-II/III sites (Figure 5A, ΔG29 = −6.7 kcal/mol) than in the four-carboxylate EF-I/IV sites (Figure 5B, ΔG29 = −5.1 kcal/mol). Protein S Binding Sites. In addition to Ca2+-binding sites in EF-hand motifs, we also examined Ca2+-binding sites in nonEF-hand-motifs, which are present in proteins such as the signaling protein (protein S) from Myxococcus xanthus. The 1.80 Å X-ray structure of protein S (PDB entry 1NPS) shows two distinct mononuclear Ca2+-binding sites consisting of only neutral ligands: In site 1, Ca2+ is heptacoordinated to the Ser and Asn side chain O, a backbone carbonyl O, and four water molecules (Figure 6A). Site 2 differs from site 1 in that one of the water molecules is replaced by a backbone carbonyl O (Figure 6B). In these complexes containing
