Electrostatic contributions to the binding of Ca2+ in calbindin D9k

A set of accurate experimental data is provided for Ca2+ ion binding to calbindin D9k, a protein in the calmodulin superfamily of intracellular regulatory proteins. The study comprises both the role of protein surface charges and the effects of added electrolyte. The two macroscopic Ca2(+)-binding constants K1 and K2 are determined for the wild-type and eight mutant calbindins in 0, 0.05, 0.10, and 0.15 M KCl from titrations in the presence of Quin 2 or 5,5'-Br2BAPTA. The mutations involve replacement of surface carboxylates (of Glu17, Asp19, Glu26, and Glu60) with the corresponding amides. It is found that K1K2 may decrease by a factor of up to 2.5 x 10(5) (triple mutant in 0.15 M KCl as compared to the wild-type protein in 0 M KCl). Ca2(+)-binding constants of the individual Ca2+ sites (microscopic binding constants) have also been determined. The positive cooperativity of Ca2+ binding, previously observed at low salt concentration [Linse et al. (1987) Biochemistry 26, 6723-6735], is also present at physiological ionic strength and amounts to 5 kJ.mol-1 at 0.15 M KCl. The electrolyte concentration and some of the mutations are found to affect the cooperativity. 39K NMR studies show that K+ binds weakly to calbindin. Two-dimensional 1H NMR studies show, however, that potassium binding does not change the protein conformation, and the large effect of KCl on the Ca2+ affinity is thus of unspecific nature. Two-dimensional 1H NMR has also been used to assess the structural consequences of the mutations through assignments of the backbone NH and C alpha H resonances of six mutants.(ABSTRACT TRUNCATED AT 250 WORDS)     

Molecular basis for co-operativity in Ca2+ binding to calbindin D9k. 1H nuclear magnetic resonance studies of (Cd2+)1-bovine calbindin D9k

The molecular basis for the co-operativity in binding of calcium ions by bovine calbindin D9k has been addressed by carrying out a comparative analysis of the solution conformation and dynamics of the apo, half saturated and fully saturated species using two-dimensional 1H nuclear magnetic resonance spectroscopy. Since the half saturated calcium form of the protein is not significantly populated under equilibrium conditions due to the co-operativity in binding of calcium ions, the half saturated cadmium form of the protein has been substituted for the calcium form. To verify that cadmium forms of calbindin D9k represent viable models for the calcium-bound species, the fully saturated cadmium form has been prepared and compared to the calcium-saturated protein. Virtually complete 1H resonance assignments have been obtained for both the (Cd2+)1 and the (Cd2+)2 states. Secondary structure elements and the global folding pattern were determined from nuclear Overhauser effects, backbone spin-spin coupling constants and slowly exchanging amide protons. Comparisons of the half saturated protein with the apo and calcium-saturated forms of calbindin D9k show that all three structures are highly similar. However, a change in the structural and dynamic properties of the protein does occur upon binding of the first ion; the half saturated form is found to be more similar to the calcium-saturated form than to the apo form. These results have important implications concerning the molecular basis for the co-operativity, and suggest that entropic effects associated with the protein dynamics play an important role.     

Structure-function relationships in EF-hand Ca2+-binding proteins. Protein engineering and biophysical studies of calbindin D9k

Genes encoding the minor A component of bovine calbindins D9k--the smallest protein known with a pair of EF-hand calcium-binding sites--with amino acid substitutions and/or deletions have been synthesized and expressed in Escherichia coli and characterized with different biophysical techniques. The mutations are confined to the N-terminal Ca2+-binding site and constitute Pro-20----Gly (M1), Pro-20----Gly and Asn-21 deleted (M2), Pro-20 deleted (M3), and Tyr-13----Phe (M4). 1H, 43Ca, and 113Cd NMR studies show that the structural changes induced are primarily localized in the modified region, with hardly any effects on the C-terminal Ca2+-binding site. The Ca2+ exchange rate for the N-terminal site changes from 3 s-1 in the wild-type protein (M0) and M4 to 5000 s-1 in M2 and M3, whereas there is no detectable variation in the Ca2+ exchange from the C-terminal site. The macroscopic Ca2+-binding constants have been obtained from equilibration in the presence of the fluorescent chelator 2-[[2-[bis(carboxymethyl)-amino]- 5-methylphenoxy]methyl]-6-methoxy-8-[bis(carboxymethyl)amino]quinoline or by using a Ca2+-selective electrode. The Ca2+ affinity of M4 was similar to that of M0, whereas the largest differences were found for the second stoichiometric step in M2 and M3. Microcalorimetric data show that the enthalpy of Ca2+ binding is negative (-8 to -13 kJ.mol-1) for all sites except the N-terminal site in M2 and M3 (+5 kJ.mol-1). The binding entropy is strongly positive in all cases. Cooperative Ca2+ binding in M0 and M4 was established through the values of the macroscopic Ca2+-binding constants. Through the observed changes in the 1H NMR spectra during Ca2+ titrations we could obtain ratios between site binding constants in M0 and M4. These ratios in combination with the macroscopic binding constants yielded the interaction free energy between the sites delta delta G as -5.1 +/- 0.4 kJ.mol-1 (M0) and less than -3.9 kJ.mol-1 (M4). There is evidence (from 113Cd NMR) for site-site interactions also in M1, M2, and M3, but the magnitude of delta delta G could not be determined because of sequential Ca2+ binding.     

Functional properties of calbindin D9K mutants with exchanged Ca2+ binding sites

We have constructed three different engineered proteins based on calbindin D9K by either exchanging the two calcium binding sites within the protein or making the amino acid sequence of the two calcium binding sites identical. The individual calcium binding sites of the engineered proteins retain most of their ion binding characteristics as well as the basal structure of their Ca2+ ligand sphere in the new environment. Even the protein with its sites interchanged, a mutation involving 30 amino acids out of a total of 75, still binds calcium with an affinity as high as that of many natural EF-hand proteins.     

Site-site communication in the EF-hand Ca2+-binding protein calbindin D9k

The cooperative binding of Ca2+ ions is an essential functional property of the EF-hand family of Ca2+-binding proteins. To understand how these proteins function, it is essential to characterize intermediate binding states in addition to the apo- and holo-proteins. The three-dimensional solution structure and fast time scale internal motional dynamics of the backbone have been determined for the half-saturated state of the N56A mutant of calbindin D9k with Ca2+ bound only in the N-terminal site. The extent of conformational reorganization and a loss of flexibility in the C-terminal EF-hand upon binding of an ion in the N-terminal EF-hand provide clear evidence of the importance of site-site interactions in this family of proteins, and demonstrates the strength of long-range effects in the cooperative EF-hand Ca2+-binding domain.     

Ca2+ binding to calbindin D9k strongly affects backbone dynamics: measurements of exchange rates of individual amide protons using 1H NMR

One- and two-dimensional 1H NMR have been used to study the backbone dynamics in Ca2(+)-free (apo) and Ca2(+)-loaded (Ca2) calbindin D9k at pH 7.5 and 25 degrees C. Hydrogen exchange rates of all 71 backbone amide protons (NH's) have been measured for the Ca2 form by both a direct exchange-out experiment and another experiment that measures the transfer of saturation from water protons to amide protons. A large number of NH's are found to be highly protected against exchange with solvent protons. The results for the Ca2 form are related to solvent accessibility and hydrogen bonding obtained in molecular dynamics simulations of calcium-loaded calbindin. The correlation with these parameters is strong within the N-terminal half of calbindin, which is found to be more stable than the C-terminal half. The amide proton exchange in the apo form is much faster than in the Ca2 form and was studied in a series of experiments in which the exchange was quenched after different times by Ca2+ addition. This experiment is applicable to all amide hydrogens that exchange slowly in the Ca2 form. For these NH's the effects of Ca2+ removal span from a 10(2)-fold decrease to a 10(5)-fold increase of the exchange rate, and the average is a 220-fold increase. The effects on individual NH exchange rates show that the four alpha-helices are almost intact after calcium removal and that the changes in dynamics involve not only the Ca2(+)-binding region. Hydrogen bonds involving backbone NH's in the Ca2+ loops appear to be broken or weakened when calbindin releases Ca2+, whereas the beta-sheet between the Ca2+ loops is found to be present in both the Ca2 and apo forms. Large Ca2(+)-induced effects on NH exchange rates were measured for a few residues at alpha-helix ends far from the two Ca2(+)-binding sites. This may be the result of a change in interhelix angles (or the rate of interhelix angle fluctuations) on calcium binding.     

The dynamics of Ca2+ ions within the solvation shell of calbindin D9k

The encounter of a Ca(2+) ion with a protein and its subsequent binding to specific binding sites is an intricate process that cannot be fully elucidated from experimental observations. We have applied Molecular Dynamics to study this process with atomistic details, using Calbindin D9k (CaB) as a model protein. The simulations show that in most of the time the Ca(2+) ion spends within the Debye radius of CaB, it is being detained at the 1st and 2nd solvation shells. While being detained near the protein, the diffusion coefficient of the ion is significantly reduced. However, due to the relatively long period of detainment, the ion can scan an appreciable surface of the protein. The enhanced propagation of the ion on the surface has a functional role: significantly increasing the ability of the ion to scan the protein's surface before being dispersed to the bulk. The contribution of this mechanism to Ca(2+) binding becomes significant at low ion concentrations, where the intervals between successive encounters with the protein are getting longer. The efficiency of the surface diffusion is affected by the distribution of charges on the protein's surface. Comparison of the Ca(2+) binding dynamics in CaB and its E60D mutant reveals that in the wild type (WT) protein the carboxylate of E60 function as a preferred landing-site for the Ca(2+) arriving from the bulk, followed by delivering it to the final binding site. Replacement of the glutamate by aspartate significantly reduced the ability to transfer Ca(2+) ions from D60 to the final binding site, explaining the observed decrement in the affinity of the mutated protein to Ca(2+).     

Nature of the individual Ca2+ binding sites in Ca2+-regenerated bacteriorhodopsin

The binding constants, K₁ and K₂, and the number of Ca²⁺ ions in each of the two high affinity sites of Ca²⁺-regenerated bacteriorhodopsin (bR) are determined potentiometrically at different pH values in the range of pH 3.5-4.5 by using the Scatchard plot method. From the pH dependence of K₁ and K₂, it was found that two hydrogen ions are released for each Ca²⁺ bound to each of the two high affinity sites. Furthermore, we have measured by a direct spectroscopic method the association constant, K_s, for the binding of Ca²⁺ to deionized bR, which is responsible for producing the blue to purple color change. Comparing the value of K_s and its pH dependence with those of K₁ and K₂ showed that the site corresponding to K_s is to be identified with that of K₂. This is in agreement with the conclusion reached previously, using a different approach, which showed that it is the second Ca²⁺ that causes the blue to purple color change.

Our studies also show that in addition to the two distinct high affinity sites, there are about four to six sites with lower binding constants. These are attributed to the nonspecific binding in bR.

     

Zn2+ binding to human calbindin D(28k) and the role of histidine residues

We have studied the binding of Zn2+ to the hexa EF-hand protein, calbindin D(28k)-a strong Ca2+-binder involved in apoptosis regulation-which is highly expressed in brain tissue. By use of radioblots, isothermal titration calorimetry, and competition with a fluorescent Zn2+ chelator, we find that calbindin D(28k) binds Zn2+ to three rather strong sites with dissociation constants in the low micromolar range. Furthermore, we conclude based on spectroscopic investigations that the Zn2+-bound state is structurally distinct from the Ca2+-bound state and that the two forms are incompatible, yielding negative allosteric interaction between the zinc- and calcium-binding events. ANS titrations reveal a change in hydrophobicity upon binding Zn2+. The binding of Zn2+ is compatible with the ability of calbindin to activate myo-inositol monophosphatase, one of the known targets of calbindin. Through site-directed mutagenesis, we address the role of cysteine and histidine residues in the binding of Zn2+. Mutation of all five cysteines into serines has no effect on Zn2+-binding affinity or stoichiometry. However, mutating histidine 80 into a glutamine reduces the binding affinity of the strongest Zn2+ site, indicating that this residue is involved in coordinating the Zn2+ ion in this site. Mutating histidines 5, 22, or 114 has significantly smaller effects on Zn2+-binding affinity.     

Protein solution structure calculations in solution: Solvated molecular dynamics refinement of calbindin D9k

The three-dimensional solution structures of proteins determinedwith NMR-derived constraints are almost always calculated in vacuo. Thesolution structure of (Ca²⁺)_{_2}-calbindinD_9k has been redetermined by new restrained molecular dynamics(MD) calculations that include Ca²⁺ ions and explicit solventmolecules. Four parallel sets of MD refinements were run to provide accuratecomparisons of structures produced in vacuo, in vacuo withCa²⁺ ions, and with two different protocols in a solvent bathwith Ca²⁺ ions. The structural ensembles were analyzed interms of structural definition, molecular energies, packing density,solvent-accessible surface, hydrogen bonds, and the coordination of calciumions in the two binding loops. Refinement including Ca²⁺ ionsand explicit solvent results in significant improvements in the precisionand accuracy of the structure, particularly in the binding loops. Theseresults are consistent with results previously obtained in free MDsimulations of proteins in solution and show that the rMD refinedNMR-derived solution structures of proteins, especially metalloproteins, canbe significantly improved by these strategies.     

Disulfide bonds in homo- and heterodimers of EF-hand subdomains of calbindin D9k: stability, calcium binding, and NMR studies.

The effect of decreased protein flexibility on the stability and calcium binding properties of calbindin D9k has been addressed in studies of a disulfide bridged calbindin D9k mutant, denoted (L39C + P43M + I73C), with substitutions Leu 39-->Cys, Ile 73-->Cys, and Pro 43-->Met. Backbone 1H NMR assignments show that the disulfide bond, which forms spontaneously under air oxidation, is well accommodated. The disulfide is inserted on the opposite end of the protein molecule with respect to the calcium sites, to avoid direct interference with these sites, as confirmed by 113Cd NMR. The effect of the disulfide bond on calcium binding was assessed by titrations in the presence of a chromophoric chelator. A small but significant effect on the cooperativity was found, as well as a very modest reduction in calcium affinity. The disulfide bond increases Tm, the transition midpoint of thermal denaturation, of calcium free calbindin D9k from 85 to 95 degrees C and Cm, the urea concentration of half denaturation, from 5.3 to 8.0 M. Calbindins with one covalent bond linking the two EF-hand subdomains are equally stable regardless if the covalent link is the 43-44 peptide bond or the disulfide bond. Kinetic remixing experiments show that separated CNBr fragments of (L39C + P43M + I73C), each comprising one EF-hand, form disulfide linked homodimers. Each homodimer binds two calcium ions with positive co-operativity, and an average affinity of 10(6) M-1. Disulfide linkage dramatically increases the stability of each homodimer. For the homodimer of the C-terminal fragment Tm increases from 59 +/- 2 without covalent linkage to 91 +/- 2 degrees C with disulfide, and Cm from approximately 1.5 to 7.5 M. The overall topology of this homodimer is derived from 1H NMR assignments and a few key NOEs.     

Paramagnetism-based versus classical constraints: An analysis of the solution structure of Ca Ln calbindin D9k

The relative importance of paramagnetism-based constraints (i.e. pseudocontact shifts, residual dipolar couplings and nuclear relaxation enhancements) with respect to classical constraints in solution structure determinations of paramagnetic metalloproteins has been addressed. The protein selected for the study is a calcium binding protein, calbindin D9k, in which one of the two calcium ions is substituted with cerium(III). From 1823 NOEs, 191 dihedral angles, 15 hydrogen bonds, 769 pseudocontact shifts, 64 orientational constraints, 26 longitudinal relaxation rates, plus 969 pseudocontact shifts from other lanthanides, a final family with backbone r.m.s.d. from the average of 0.25 A was obtained. Then, several families of structures were generated either by removing subsets of paramagnetism-based constraints or by removing increasing numbers of NOEs. The results show the relative importance of the various paramagnetism-based constraints and their good complementarity with the diamagnetic ones. Although a resolved structure cannot be obtained with paramagnetism-based constraints only, it is shown that a reasonably well resolved backbone fold can be safely obtained by retaining as few as 29 randomly chosen long-range NOEs using the standard version of the program PSEUDYANA.     

Tb3+ binding to Ca2+ and Mg2+ binding sites on sarcoplasmic reticulum ATPase

The interactions of Tb3+ and sarcoplasmic reticulum (SR) were investigated by inhibition of Ca2+-activated ATPase activity and enhancement of Tb3+ fluorescence. Ca2+ protected against Tb3+ inhibition of SR ATPase activity. The apparent association constant for Ca2+, determined from the protection, was about 6 x 10(6) M-1, suggesting that Tb3+ inhibits the ATPase activity by binding to the high affinity Ca2+ binding sites. Mg2+ did not protect in the 2-20 mM range. The association constant for Tb3+ binding to this Ca2+ site was estimated to be about 1 x 10(9) M-1. No cooperativity was observed for Tb3+ binding. No enhancement of Tb3+ fluorescence was detected. A second group of binding sites, with weaker affinity for Tb3+, was observed by monitoring the enhancement of Tb3+ fluorescence (lambda ex 285 nm, lambda em 545 nm). The fluorescence intensity increased 950-fold due to binding. Ca2+ did not complete for binding at these sites, but Mg2+ did. The association constant for Mg2+ binding was 94 M-1, suggesting that this may be the site that catalyzes phosphorylation of the ATPase by inorganic phosphate. For vesicles, Tb3+ binding to these Mg2+ sites was best described as binding to two classes of binding sites with negative cooperativity. If the SR ATPase was solubilized in the nonionic detergent C12E9 (dodecyl nonaoxyethylene ether alcohol), in the absence of Ca2+, only one class of Tb3+ binding sites was observed. The total number of sites appeared to remain constant. If Ca2+ was included in the solubilization step, Tb3+ binding to these Mg2+ binding sites displayed positive cooperativity (Hill coefficient, 2.1). In all cases, the apparent association constant for Tb3+, in the presence of 5 mM MgCl2, was in the range of 1-5 x 10(4) M-1.     

Specificity of ligand binding to transport sites: Ca2+ binding to the Ca2+ transport ATPase and its dependence on H+ and Mg2+

Ligand binding to transport sites constitutes the initial step in the catalytic cycle of transport ATPases. Here, we consider the well characterized Ca²⁺ ATPase of sarcoplasmic reticulum (SERCA) and describe a series of Ca²⁺ binding isotherms obtained by equilibrium measurements in the presence of various H⁺ and Mg²⁺ concentrations. We subject the isotherms to statistical mechanics analysis, using a model based on a minimal number of mechanistic steps. The analysis allows satisfactory fits and yields information on occupancy of the specific Ca²⁺ sites under various conditions. It also provides a fundamental method for analysis of binding specificity to transport sites under equilibrium conditions that lead to tightly coupled catalytic activation.     

pK(a) calculations of calbindin D(9k): effects of Ca(2+) binding, protein dielectric constant, and ionic strength

Calbindin is a small (75 residues) helix-loop-helix ("EF-hand") calcium-binding protein belonging to the calmodulin superfamily. It binds two Ca(2+) ions. Continuum electrostatics in combination with the boundary element method was employed for the calculation of the acid-dissociation constants K(a) (pK(a) = -log K(a)) values of all titratable residues in the protein. The objectives were to determine quantitatively the effects of divalent ion binding and small ion-induced structural changes on predicted pK(a)'s. Computations were carried out for the apo and holo form of calbindin, for which both X-ray and NMR structures were available. Comparison was made with several sets of experimental pK(a) values determined by NMR spectroscopy. Different choices of the dielectric constant (ranging from 4 to 78.5) for calbindin and variations in ionic strength (from 0 to 0.3 M) were investigated in a systematic fashion. Removal of the two bound Ca(2+) ions increases the pK(a) values of all residues if no conformational changes were allowed. If conformational differences between the apo and holo were accounted for, shifts in either direction were observed. Titrating groups that are directly involved in Ca(2+) binding (Asp and Glu) required a dielectric constant of 78.5 for the holo structure to obtain a reasonable estimate of their pK(a)'s. For the apo structure, passable values for the pK(a)'s of these ligating groups could be determined if the structure was allowed to relax upon ion removal.     

Effects of calcium binding on the side-chain methyl dynamics of calbindin D9k: a 2H NMR relaxation study

The effects of Ca(2+) binding on the side-chain methyl dynamics of calbindin D(9k) have been characterized by (2)H NMR relaxation rate measurements. Longitudinal, transverse in-phase, quadrupolar order, transverse anti-phase and double quantum relaxation rates are reported for both the apo and Ca(2+)-loaded states of the protein at two magnetic field strengths. The relatively large size of the data set allows for a detailed analysis of the underlying conformational dynamics by spectral density mapping and model-free fitting procedures. The results reveal a correlation between a methyl group's distance from the Ca(2+) binding sites and its conformational dynamics. Several methyl groups segregate into two limiting classes, one proximal and the other distal to the binding sites. Methyl groups in these two classes respond differently to Ca(2+) binding, both in terms of the timescale and amplitude of their fluctuations. Ca(2+) binding elicits a partial immobilization among methyl groups in the proximal class, which is consistent with previous studies of calbindin's backbone dynamics. The distal class, however, exhibits a trend that could not be inferred from the backbone data in that its mobility actually increases with Ca(2+) binding. We have introduced the term polar dynamics to describe this type of organization across the molecule. The trend may represent an important mechanism by which calbindin D(9k) achieves high affinity binding while minimizing the corresponding loss of conformational entropy.     

Ion-binding properties of calbindin D9k: a Monte Carlo simulation study

Monte Carlo simulations are used to calculate the binding constant of two Ca2+ ions to the protein bovine calbindin D9k. The change in binding constant with respect to mutation of charged amino acids, presence of various electrolytes, protein concentration, solution pH, and competitive binding of monovalent ions is investigated. Each of these factors may have a large influence on the binding constant. The simulations are performed in a dielectric continuum model, the so-called primitive model of electrolyte theory, with a fixed protein structure and a uniform dielectric permittivity. The calculated binding constants are in excellent agreement with experimental data and describe changes in the binding constant over six orders of magnitude.     

Ionization behavior of acidic residues in calbindin D(9k).

The ionization state of seven glutamate residues, one aspartate, and the C-terminal alpha-COOH group in bovine apo calbindin D(9k) has been studied by measurement and modeling of the pH titration curves and apparent pK(a) values. The observed pK(a) ranged from 3.0 to 6.5. Most of the observed acidic groups were half-ionized at lower pH values than those in unstructured proteins. As a rule, the ionization equilibria extended over a wider pH range than in the case of unperturbed single titrations, indicating a complex influence of protein charges on the charge state of each individual residue. Glu17, which is a backbone Ca(2+)-ligand in the N-terminal binding loop of calbindin D(9k), was half-protonated at pH 3.6 but manifested biphasic titration with apparent pK(a) values of 3.2 and 6.5. Complementary Monte Carlo simulations of the titration process and pK(a) values of the acidic groups in calbindin D(9k) reproduce the experimentally observed titration features, except for the pronounced double titration of Glu17. Discrepancies between the results from direct measurement and from modeling may be partly caused by changes in the protein structure when the net charge changes from -8 to +11 over the isoelectric point at pH 5. Proteins 1999;37:106-115.     

Biophysical studies of engineered mutant proteins based on calbindin D9k modified in the pseudo EF-hand

The genes for four mutant proteins from calbindin D9k, all with mutations in the N-terminal Ca2+-binding domain (pseudo EF-hand) have been synthesized and expressed in Escherichia coli. The purification scheme has been modified to minimize the formation of deamidated proteins. The set of modifications in the pseudo EF-hand is an attempt to turn this site into a structure resembling an archetypal EF-hand, with its characteristic 113Cd-NMR shift (-80 to -110 ppm) and high calcium-binding constants, whereas the C-terminal Ca2(+)-binding site (EF-hand) is kept intact in all mutant proteins. The mutant proteins studied here all have pseudo EF-hands with a lower calcium-binding constant and a higher calcium off-rate to the pseudo EF-hand than the wild-type protein. From the results obtained it is obvious that proline 20 in the pseudo EF-hand, which has been deleted or replaced by glycine in three of the mutants, has a stabilizing effect on calcium binding to that site. Furthermore, the modifications in the pseudo EF-hand seem to have only a local effect, leaving the tertiary structure of the protein and the calcium-binding properties of the unmodified site virtually unchanged.