Rat alpha- and beta-parvalbumins: comparison of their pentacarboxylate and site-interconversion variants

Introduction of a fifth carboxylate into the ligand array of the CD site (via the combined S55D and E59D mutations) or the EF site (G98D) of rat alpha-parvalbumin substantially increases divalent ion affinity. This behavior, in conflict with that seen in model peptide systems, agrees with existing data for rat beta-parvalbumin [Henzl et al. (1996) Biochemistry 35, 5856-5869]. The complete analysis of the S55D/E59D double variant necessitated characterization of alpha E59D. Whereas the D59E mutation has minimal influence on beta CD site affinity, E59D has a major impact on the alpha CD site, lowering the apparent association constant by a factor of 14. The thermodynamic consequences of exchanging the rat alpha CD and EF site ligand arrays, which differ at the +z and -x coordination positions, were also examined. When the alpha CD array is imported into the EF site, it acquires a low-affinity phenotype, in agreement with previous findings for beta [Henzl et al. (1998) Biochemistry 37, 9101-9111]. However, when the EF ligand array is introduced into the alpha CD binding loop, it retains a high-affinity signature. This result, contrary to that observed in beta, suggests that the influence of the parvalbumin CD site environment supersedes the intrinsic behavior of the ligand array, a conclusion further supported by the disparate impact of the beta D59E and alpha E59D mutations.     

Influence of monovalent cation identity on parvalbumin divalent ion-binding properties

Rat alpha- and beta-parvalbumins have distinct monovalent cation-binding properties [Henzl et al. (2000) Biochemistry 39, 5859-5867]. Beta binds two Na(+) or one K(+), and alpha binds one Na(+) and no K(+). Ca(2+) abolishes these binding events, suggesting that the monovalent ions occupy the EF-hand motifs. This study compares alpha and beta divalent ion affinities in Na(+) and K(+) solutions. Solvent cation identity seriously affects alpha. In Hepes-buffered NaCl, at 5 degrees C, the macroscopic Ca(2+)-binding constants are 2.6 x 10(8) and 6.4 x 10(7) M(-1) and the Mg(2+) constants, 1.8 x 10(4) and 4.3 x 10(3) M(-1). In Hepes-buffered KCl, the Ca(2+) values increase to 2.9 x 10(9) and 6.6 x 10(8) M(-1) and the Mg(2+) values to 2.2 x 10(5) and 3.7 x 10(4) M(-1). Monte Carlo simulation of alpha binding data-employing site-specific constants and explicitly considering Na(+) binding-yields a K(Na) of 630 M(-1) and indicates that divalent ion-binding is positively cooperative. NMR data suggest that the lone Na(+) ion occupies the CD loop. Solvent cation identity has a smaller impact on beta. In Na(+), the Ca(2+) constants for the EF and CD sites are 2.3 x 10(7) and 1.5 x 10(6) M(-1), respectively; the Mg(2+) constants are 9.2 x 10(3) and 1.7 x 10(2) M(-1). In K(+), these values shift to 3.1 x 10(7) and 3.8 x 10(6) M(-1) and the latter to 1.4 x 10(4) and 2.9 x 10(2) M(-1). These data suggest that parvalbumin divalent ion affinity, particularly that of rat alpha, can be significantly attenuated by increased intracellular Na(+) levels.     

Estimation of parvalbumin Ca(2+)- and Mg(2+)-binding constants by global least-squares analysis of isothermal titration calorimetry data

The use of competitive isothermal titration calorimetry (ITC) to measure high-affinity binding constants has been largely restricted to systems with a single binding site or multiple identical sites. This study demonstrates the extension of this approach to proteins with two nonequivalent EF-hand Ca(2+)-binding sites--rat beta parvalbumin and the S55D/E59D variant of rat alpha parvalbumin. The method involves simultaneous (global) least-squares analysis of titrations with Ca(2+), with Mg(2+), with Ca(2+) in the presence of Mg(2+), and with Ca(2+) or Mg(2+) in the presence of a competitive chelator (EDTA or EGTA). The Ca(2+) and Mg(2+) binding constants obtained for rat beta agree well with estimates obtained by flow dialysis. Although the Ca(2+) affinity of alpha S55D/E59D is too high to measure by flow dialysis, it was amenable to analysis using the ITC-based approach. The combined S55D and E59D mutations increase the Ca(2+) and Mg(2+) affinities of the mutated binding site by factors of 14 and 26, respectively. This behavior is consistent with that seen previously for the rat beta S55D variant.     

Crystal structure of the D94S/G98E variant of rat alpha-parvalbumin. An explanation for the reduced divalent ion affinity

Simultaneous replacement of Asp-94 with serine and Gly-98 with glutamate in rat alpha-parvalbumin creates a CD-site ligand array in the context of the EF-site binding loop. Previous work has shown that, relative to the wild-type CD site, this engineered site has markedly reduced Ca(2+) affinity. Seeking an explanation for this phenomenon, we have obtained the crystal structure of the alpha D94S/G98E variant. The Ca(2+) coordination within the engineered EF site of the 94/98E variant is nearly identical to that within the CD site, suggesting that the attenuated affinity of the EF site in 94/98E is not a consequence of suboptimal coordination geometry. We have also examined the divalent ion binding properties of the alpha 94/98E variant in both Na(+)- and K(+)-containing buffers. Although the Ca(2+) and Mg(2+) affinities are higher in K(+) solution, the increases are comparable to those observed for wild-type alpha. Consistent with that finding, the apparent Na(+) stoichiometry, estimated from stability studies conducted as a function of Na(+) concentration, is 1.0 +/- 0.1, identical to that of wild-type alpha. Thus, the reduced affinity for divalent ions is evidently not the result of heightened monovalent ion competition. The thermodynamic analysis indicates that the less favorable Gibbs free energy of binding reflects a substantial enthalpic penalty. Significantly, the crystal structure reveals a steric clash between Phe-57 and the C(gamma) atom of Glu-98. The consequent displacement of Phe-57 also produces a close contact with Ser-55. Thus, steric interference may be the source of the enthalpic penalty.     

Calcium-binding properties of wild-type and EF-hand mutants of S100B in the presence and absence of a peptide derived from the C-terminal negative regulatory domain of p53

S100B is a dimeric Ca(2+)-binding protein that undergoes a 90 +/- 3 degrees rotation of helix 3 in the typical EF-hand domain (EF2) upon the addition of calcium. The large reorientation of this helix is a prerequisite for the interaction between each subunit of S100B and target proteins such as the tumor suppressor protein, p53. In this study, Tb(3+) was used as a probe to examine how binding of a 22-residue peptide derived from the C-terminal regulatory domain of p53 affects the rate of Ca(2+) ion dissociation. In competition studies with Tb(3+), the dissociation rates of Ca(2+) (k(off)) from the EF2 domains of S100B in the absence and presence of the p53 peptide was determined to be 60 and 7 s(-)(1), respectively. These data are consistent with a previously reported result, which showed that that target peptide binding to S100B enhances its calcium-binding affinity [Rustandi et al. (1998) Biochemistry 37, 1951-1960]. The corresponding Ca(2+) association rate constants for S100B, k(on), for the EF2 domains in the absence and presence of the p53 peptide are 1.1 x 10(6) and 3.5 x 10(5) M(-)(1) s(-)(1), respectively. These two association rate constants are significantly below the diffusion control ( approximately 10(9) M(-)(1) s(-)(1)) and likely involve both Ca(2+) ion association and a Ca(2+)-dependent structural rearrangement, which is slightly different when the target peptide is present. EF-hand calcium-binding mutants of S100B were engineered at the -Z position (EF-hand 1, E31A; EF-hand 2, E72A; both EF-hands, E31A + E72A) and examined to further understand how specific residues contribute to calcium binding in S100B in the absence and presence of the p53 peptide.     

Effects of mutations in the calcium-binding sites of recoverin on its calcium affinity: evidence for successive filling of the calcium binding sites

A molecule of the photoreceptor Ca(2+)-binding protein recoverin contains four potential EF-hand Ca(2+)-binding sites, of which only two, the second and the third, are capable of binding calcium ions. We have studied the effects of substitutions in the second, third and fourth EF-hand sites of recoverin on its Ca(2+)-binding properties and some other characteristics, using intrinsic fluorescence, circular dichroism spectroscopy and differential scanning microcalorimetry. The interaction of the two operating binding sites of wild-type recoverin with calcium increases the protein's thermal stability, but makes the environment around the tryptophan residues more flexible. The amino acid substitution in the EF-hand 3 (E121Q) totally abolishes the high calcium affinity of recoverin, while the mutation in the EF-hand 2 (E85Q) causes only a moderate decrease in calcium binding. Based on this evidence, we suggest that the binding of calcium ions to recoverin is a sequential process with the EF-hand 3 being filled first. Estimation of Ca(2+)-binding constants according to the sequential binding scheme gave the values 3.7 x 10(6) and 3.1 x 10(5) M(-1) for third and second EF-hands, respectively. The substitutions in the EF-hand 2 or 3 (or in both the sites simultaneously) do not disturb significantly either tertiary or secondary structure of the apo-protein. Amino acid substitutions, which have been designed to restore the calcium affinity of the EF-hand 4 (G160D, K161E, K162N, D165G and K166Q), increase the calcium capacity and affinity of recoverin but also perturb the protein structure and decrease the thermostability of its apo-form.     

Solution structure of Ca2+-free rat alpha-parvalbumin

Mammals express two parvalbumins-an alpha isoform and a beta isoform. In rat, the alpha-parvalbumin (alpha-PV) exhibits superior divalent ion affinity. For example, the standard free energies for Ca2+ binding differ by 5.5 kcal/mol in 0.15 M KCl (pH 7.4). High-resolution structures of the Ca2+-bound proteins provide little insight into this disparity, prompting a structural analysis of the apo-proteins. A recent analysis of rat beta-PV suggested that Ca2+ removal provokes substantial conformational changes-reorientation of the C, D, and E helices; reorganization of the hydrophobic core; reduced interdomain contact; and remodeling of the AB domain. The energetic penalty attendant to reversing these changes, it was suggested, could contribute to the attenuated divalent ion-binding signature of that protein. That hypothesis is supported by data presented herein, describing the solution structure and peptide backbone dynamics of Ca2+-free rat alpha-PV. In marked contrast to rat beta-PV, the apo- and Ca2+-loaded forms of the rat alpha isoform are quite similar. Significant structural differences appear to be confined to the loop regions of the molecule. This finding implies that the alpha-PV isoform enjoys elevated divalent ion affinity because the metal ion-binding events do not require major structural rearrangement and the concomitant sacrifice of binding energy.     

Site-specific substitution of glutamate for aspartate at position 59 of rat oncomodulin

Replacement of the aspartate residue at position 59 of rat oncomodulin by glutamate by oligonucleotide-directed mutagenesis has afforded a protein which more closely resembles rat parvalbumin, at least judged by its interaction with the luminescent lanthanide ion Eu3+. The single-peak 7F0----5D0 spectrum observed at pH 5.0 with the fully bound wild-type protein is replaced by one which clearly shows two features at 5791 and 5796 A, arising from Eu3+ ions bound at the CD and EF sites, respectively. Furthermore, the pH dependence of the spectrum is substantially altered; the pKa observed for the CD domain, in which aspartate 59 residues, is shifted upward from pH 6.0 for the wild-type recombinant protein to pH 6.8 in the D59E mutant. Moreover, the maximum in the high-pH spectrum is shifted from 5781 to 5784 A. All three changes are indicative of a CD binding domain having increased parvalbumin-like character. Interestingly, however, the D59E substitution has only a modest effect on the Ca2+- and Mg2+-binding properties of the CD domain. For the wild-type protein, KCa = 7.8 x 10(-7) M and KMg = 3 x 10(-3) M. These affinities are more than an order of magnitude weaker than those seen for various parvalbumins and substantiate previous claims for calcium specificity made for the oncomodulin CD domain. Replacement of aspartate 59 by glutamate resulted in minor increases in affinity of the CD domain for Ca2+ (KCa = 5.5 x 10(-7) M) and Mg2+ (KMg = 1 x 10(-3) M). These findings strongly suggest that residues in oncomodulin besides aspartate 59 are important determinants of the observed calcium specificity of the CD calcium-binding domain. The consequences of the substitution at residue 59 appear to be confined to the CD domain. For the EF site in wild-type recombinant oncomodulin, KCa = 4.2 x 10(-8) M and KMg = 1.6 x 10(-4) M. The corresponding values for the D59E site-specific variant are identical within experimental error (KCa = 4.2 x 10(-8) M and KMg = 1.8 x 10(-4) M).     

Potential influence of Asp in the Ca2+ coordination position 5 of parvalbumin on the calcium-binding affinity: a computational study

Parvalbumins (PV) are calcium-binding proteins, all sharing the common helix-loop-helix (EF-hand) motif. This motif contains a central twelve-residue Ca(2+)-binding loop with the flanking helices positioned roughly perpendicular to each other. The precise role of these coordination residues has been the subject of intense studies. In this work, we focus on the coordination position 5 in the CD Ca(2+)-binding site of silver hake parvalbumin isoform B (SHPV-B). The most common residue at site 5 of calcium-binding loop in canonical EF-hands is Asp [B.J. Marsden, G.S. Shaw, B.D. Sykes, Biochem. Cell Biol. 68 (1990) 587-601], but in the CD site of PV, this position is almost always serine (Ser). The substitution of Ser with Asp will add the 5th carboxylate residue in the CD coordination sphere. However, as predicted by the acid pair hypothesis, the Ca(2+)-binding affinity would be maximized in an EF-hand motif that has four carboxylate ligands paired along the +/-x, and +/-z-axes [R.E. Reid, R.S. Hodges, J. Theor. Biol. 84 (1980) 401-444]. Molecular dynamics simulations and free energy calculations were employed to investigate the influence of Ser to Asp mutation at position 5 on calcium-binding affinity. We found that the Asp variant exhibited remarkable stability during the entire molecular dynamics simulation, with not only the retention of the Ca(2+)-binding site, but also increased compactness in the coordination sphere. The S55D fragment also accommodated Ca(2+) well. We conclude that the reason why Asp which is the most common residue at site 5 of calcium-binding loop in canonical EF-hands has never been identified at this position experimentally for PVs might be related to its physiological functions.     

Recoverin is a zinc-binding protein

Recoverin is an N-myristoylated 23 kDa calcium-binding protein from retina, which modulates the Ca2+-sensitive deactivation of rhodopsin via Ca2+-dependent inhibition of rhodopsin kinase. It was shown by intrinsic and bis-ANS probe fluorescence, circular dichroism, and differential scanning calorimetry that myristoylated recombinant recoverin interacts specifically with zinc ions. Similar to the calcium binding, the binding of zinc to Ca2+-loaded recoverin additionally increases its alpha-helical content, hydrophobic surface area, and environmental mobility/polarity of its tryptophan residues. In contrast to the calcium binding, the binding of zinc decreases thermal stability of the Ca2+-loaded protein. Zn2+-titration of recoverin, traced by bis-ANS fluorescence, reveals binding of a single Zn2+ ion per protein molecule. It was shown that the double-mutant E85Q/E121Q with inactivated Ca2+-binding EF-hands 2 and 3 (Alekseev, A. M.; Shulga-Morskoy, S. V.; Zinchenko, D. V.; Shulga-Morskaya, S. A.; Suchkov, D. V.; Vaganova, S. A.; Senin, I. I.; Zargarov, A. A.; Lipkin, V. M.; Akhtar, M.; Philippov, P. P. FEBS Lett. 1998, 440, 116-118), which can be considered as an analogue of the apo-protein, binds Zn2+ ion as well. Apparent zinc equilibrium binding constants evaluated from spectrofluorimetric Zn2+-titrations of the protein are 1.4 x 10(5) M(-1) (dissociation constant 7.1 microM) for Ca2+-loaded wild-type recoverin and 3.3 x 10(4) M(-1) (dissociation constant 30 microM) for the E85Q/E121Q mutant (analogue of apo-recoverin). Study of the binding of wild-type recoverin to ROS membranes showed a zinc-dependent increase of its affinity for the membranes, without regard to calcium content, suggesting further solvation of a protein myristoyl group upon Zn2+ binding. Possible implications of these findings to the functioning of recoverin are discussed.     

Protein surface charges and Ca2+ binding to individual sites in calbindin D9k: stopped-flow studies

The kinetics of calcium dissociation from two groups of site-specific mutants of calbindin D9k--a protein in the calmodulin superfamily with two Ca2+ sites and a tertiary structure closely similar to that of the globular domains of troponin C and calmodulin--have been studied by stopped-flow kinetic methods, using the fluorescent calcium chelator Quin 2, and by 43Ca NMR methods. The first group of mutants comprises all possible single, double, and triple neutralizations of three particular carboxylate groups (Glu-17, Asp-19, and Glu-26) that are located on the surface of the protein. These carboxylates are close to the two EF-hand calcium binding sites, but are not directly liganded to the Ca2+ ions. Conservative modification of these negative carboxylate side chains by conversion to the corresponding amides results in a marked reduction in the Ca2+ binding constants for both sites, as recently reported [Linse et al. (1988) Nature 335, 651-652]. The stopped-flow kinetic results show that this reduction in Ca2+ affinity derives primarily from a reduction in the Ca2+ association rate constant, kon. The estimated maximum value of the association rate constant (kon(max) for Ca2+ binding to the wild-type protein is ca. 10(9) M-1 s-1. In contrast, for the mutant protein with three charges neutralized the maximum association rate constant is estimated to be only 2 X 10(7) M-1 s-1.(ABSTRACT TRUNCATED AT 250 WORDS)     

Divalent cation sensitivity of BK channel activation supports the existence of three distinct binding sites

Mutational analyses have suggested that BK channels are regulated by three distinct divalent cation-dependent regulatory mechanisms arising from the cytosolic COOH terminus of the pore-forming alpha subunit. Two mechanisms account for physiological regulation of BK channels by microM Ca2+. The third may mediate physiological regulation by mM Mg2+. Mutation of five aspartate residues (5D5N) within the so-called Ca2+ bowl removes a portion of a higher affinity Ca2+ dependence, while mutation of D362A/D367A in the first RCK domain also removes some higher affinity Ca2+ dependence. Together, 5D5N and D362A/D367A remove all effects of Ca2+ up through 1 mM while E399A removes a portion of low affinity regulation by Ca2+/Mg2+. If each proposed regulatory effect involves a distinct divalent cation binding site, the divalent cation selectivity of the actual site that defines each mechanism might differ. By examination of the ability of various divalent cations to activate currents in constructs with mutationally altered regulatory mechanisms, here we show that each putative regulatory mechanism exhibits a unique sensitivity to divalent cations. Regulation mediated by the Ca2+ bowl can be activated by Ca2+ and Sr2+, while regulation defined by D362/D367 can be activated by Ca2+, Sr2+, and Cd2+. Mn2+, Co2+, and Ni2+ produce little observable effect through the high affinity regulatory mechanisms, while all six divalent cations enhance activation through the low affinity mechanism defined by residue E399. Furthermore, each type of mutation affects kinetic properties of BK channels in distinct ways. The Ca2+ bowl mainly accelerates activation of BK channels at low [Ca2+], while the D362/D367-related high affinity site influences both activation and deactivation over the range of 10-300 microM Ca2+. The major kinetic effect of the E399-related low affinity mechanism is to slow deactivation at mM Mg2+ or Ca2+. The results support the view that three distinct divalent-cation binding sites mediate regulation of BK channels.     

NMR studies of calcium-binding to mutant alpha-spectrin EF-hands

The co-operative calcium binding mechanism of the two C-terminal EF-hands of human alphaII-spectrin has been investigated by site-specific mutagenesis and multi-dimensional NMR spectroscopy. To analyse the calcium binding of each EF-hand independently, two mutant structures (E33A and D69S) of wild type alpha-spectrin were prepared. According to NMR analysis both E33A and D69S were properly folded. The unmutated EF-hand in these mutants remained nearly intact and active in calcium binding, whereas the mutated EF-hand lost its affinity for calcium completely. The apparent calcium binding affinity of the E33A mutant was much lower compared to the D39S mutant (approximately 2470 microM and approximately 240 microM, respectively). When the chemical shift perturbations were followed upon calcium titration, a positive correlation between the D69S mutant and the binding of the first calcium ion to the wild type was revealed. These observations showed that the first EF-hand in spectrin binds the first calcium ion and thereby triggers a conformational change that allows the second calcium ion to bind to the other EF-hand.     

Residues 110-126 in the A1 domain of factor VIII contain a Ca2+ binding site required for cofactor activity

Generation of factor VIII cofactor activity requires divalent metal ions such as Ca2+ or Mn2+. Evaluation of cofactor reconstitution from isolated factor VIIIa subunits revealed the presence of a functional Ca2+ binding site within the A1 subunit. Isothermal titration calorimetry demonstrated at least two Ca2+ binding sites of similar affinity (K(d) = 0.74 microm) within the A1 subunit. Mutagenesis of an acidic residue-rich region in the A1 domain (residues 110-126) homologous to a putative Ca2+ binding site in factor V (Zeibdawi, A. R., and Pryzdial, E. L. (2001) J. Biol. Chem. 276, 19929-19936) and expression of B-domainless factor VIII molecules yielded reagents to probe Ca2+ and Mn2+ binding in a functional assay. Basal activity observed for wild type factor VIII in a metal ion-free buffer was enhanced approximately 2-fold with saturating Ca2+ or Mn2+ and yielded functional K(d) values of 1.2 and 1.40 microm, respectively. Ca2+ binding affinity was greatly reduced (or lost) in several mutants including E110A, E110D, D116A, E122A, D125A, and D126A. Alternatively, E113A, D115A, and E124A showed wild type-like activity with little or no reduction in Ca2+ affinity. However, Mn2+ affinity was minimally altered except for mutant D125A (and D116A). These results are consistent with region 110-126 serving a critical role for Ca2+ coordination with selected residues capable of contributing to a partially overlapping site for Mn2+, and that occupancy of either site is required for maximal cofactor activity.     

Effects of metal-binding loop mutations on ligand binding to calcium- and integrin-binding protein 1. Evolution of the EF-hand?

Calcium- and integrin-binding protein 1 (CIB1) is a ubiquitous, multifunctional regulatory protein consisting of four helix-loop-helix EF-hand motifs. Neither EF-I nor EF-II binds divalent metal ions; however, EF-III is a mixed Mg2+/Ca2+-binding site, and EF-IV is a higher-affinity Ca2+-specific site. Through the generation of several CIB1 mutant proteins, we have investigated the importance of the last (-Z) metal-coordinating position of EF-III (D127) and EF-IV (E172) with respect to the binding of CIB1 to Mg2+, Ca2+, and its biological target, the cytoplasmic domain of the platelet alphaIIb integrin. A D127N mutant had reduced Mg2+ and Ca2+ affinity at EF-III but retained affinity for the alphaIIb domain. A D127E mutant had increased Mg2+ and Ca2+ affinity at EF-III, but unexpectedly, the affinity for the alphaIIb domain was too low for binding to be observed. E172Q and E172D mutants showed no and weak Mg2+ binding at EF-IV, respectively, and each mutant had reduced Ca2+ affinity at EF-IV and showed moderate metal-dependent differences in affinity for the alphaIIb domain. Finally, a D127Q mutant bound Mg2+ and Ca2+ in a manner similar to that of D127N, but like that of D127E, the affinity for the alphaIIb domain was reduced below the detection limit. These data, combined with a NMR-based structural comparison of the Mg2+- and Ca2+-loaded CIB1-alphaIIb peptide complexes, suggest that the D127E and D127Q mutations have a disruptive effect on alphaIIb binding since they expand the metal-binding loop and change the alpha-helix positions in EF-III. Conversely, upon replacement of the ancestral Glu with Asp at the -Z position of EF-III, CIB1 gained affinity for alphaIIb, and the Ca2+ affinity of CIB1 shifted into a range where the protein is able to act as an intracellular Ca2+ sensor.     

Divalent ion-binding properties of the two avian beta-parvalbumins

Birds express three parvalbumins, one alpha isoform and two beta isoforms. The latter are known as avian thymic hormone (ATH) and avian parvalbumin 3. Although both were discovered in thymus tissue, and presumably function in T-cell maturation, they have been detected in other tissue settings. We have conducted detailed Ca2+- and Mg2+-binding studies on recombinant ATH and the C72S variant of CPV3, employing global analysis of isothermal titration calorimetry data. In Hepes-buffered saline, ATH binds Ca2+ with apparent microscopic binding constants of 2.4 +/- 0.2 x 10(8) and 1.0 +/- 0.1 x 10(8) M(-1). The corresponding values for CPV3-C72S are substantially lower, 4.5 +/- 0.5 x 10(7) and 2.4 +/- 0.2 x 10(7) M(-1), a 1.9-kcal/mol difference in binding free energy. Thus, the beta-parvalbumin lineage displays a spectrum of Ca2+-binding affinity, with ATH and the mammalian beta isoform at the high- and low-affinity extremes and CPV3 in the middle. Interestingly, despite its decreased Ca2+ affinity, CPV3-C72S exhibits increased affinity for Mg2+, relative to ATH. Whereas the latter displays Mg2+-binding constants of 2.2 +/- 0.2 x 10(4) and 1.2 +/- 0.1 x 10(4) M(-1), CPV3-C72S yields values of 5.0 +/- 0.8 x 10(4) and 2.1 +/- 0.3 x 10(4) M(-1).     

Calbindin-D28K, a 1 alpha,25-dihydroxyvitamin D3-induced calcium-binding protein, binds five or six Ca2+ ions with high affinity

Calbindin-D28K is a 1 alpha,25-dihydroxyvitamin D3-dependent protein that belongs to the superfamily of high affinity calcium-binding proteins which includes parvalbumin, calmodulin, and troponin C. All of these proteins bind Ca2+ ligands by an alpha-helix-loop-alpha-helix domain that is termed an EF-hand. Calbindin-D28K has been reported previously to have four high affinity Ca2(+)-binding sites (KD less than 10(-7)) as quantitated by equilibrium dialysis. With the determination of the amino acid sequence, it was clear that there are in fact six apparent EF-hand domains, although the Ca2(+)-binding functionality of the two additional domains was unclear. It was of interest to quantitate the Ca2(+)-binding ability of chick intestinal calbindin-D28K utilizing several different Ca2+ titration methods that cover a range of macroscopic binding constants for weak or strong Ca2+ sites. Titrations with the Ca2+ chelator dibromo-1,2-bis(2-aminophenoxy)-ethane-N,N,N',N'-tetraacetic acid (5,5'-Br2BAPTA), a Ca2+ selective electrode, and as followed by 1H NMR, which measure KD values of 10(-6)-10(-8) M, 10(-4)-10(-7) and 10(-3)-10(-5) M, respectively, gave no evidence for the presence of weak Ca2(+)-binding sites. However, Ca2+ titration of the fluorescent Ca2+ chelator Quin 2 in the presence of calbindin-D28K yielded a least squares fit optimal for 5.7 +/- 0.8 Ca2(+)-binding sites with macroscopic dissociation constants around 10(-8) M. The binding of Ca2+ by calbindin was found to be cooperative with at least two of the sites exhibiting positive cooperativity.     

Identification of calcium binding sites that regulate potentiation of a neuronal nicotinic acetylcholine receptor.

The divalent cation calcium potentiates the physiological response of neuronal nicotinic receptors to agonists by enhancing ionic current amplitudes, apparent agonist affinity and cooperativity. Here we show that mutations in several consensus Ca2+ binding sequences from the N-terminal domain of the neuronal alpha 7 nicotinic acetylcholine receptor alter Ca2+ potentiation of the alpha 7-V201-5HT3 chimera. Mutations E18Q or E44Q abolish calcium-enhanced agonist affinity but preserve the calcium increase of plateau current amplitudes and cooperativity. On the other hand, mutations of amino acids belonging to the 12 amino acid canonical domain (alpha 7 161-172) alter all features of potentiation by enhancing (D163, S169), reducing (E161, S165, Y167) or abolishing (E172) calcium effects on ionic current amplitudes and agonist affinity. Introduction of the alpha 7 161-172 domain in the calcium insensitive 5-hydroxytryptamine (5HT3) serotoninergic receptor results in a receptor activated by 5HT and potentiated by calcium. In vitro terbium fluorescence studies with an alpha 7 160-174 peptide further show that mutation E172Q also alters in vitro calcium binding. Data are consistent with the occurrence of distinct categories of regulatory calcium binding sites, among which the highly conserved (alpha 7 161-172) domain may simultaneously contribute to calcium and agonist binding.     

Stabilizing influence of calcium and magnesium ions on the decameric structure of chiton hemocyanins

The stabilizing effects of Ca2+ and Mg2+ ions on the decameric structure of hemocyanins from two representative chitons, Stenoplax conspicua and Mopalia muscosa were investigated by light-scattering molecular weight measurements, ultracentrifugation, absorbance, and circular dichroism methods. The dissociation profiles at any given pH resulting from the decrease in divalent ion concentration, investigated at a fixed protein concentration of 0.1 g.liter-1, could be fitted by a decamer-to-dimer-to monomer scheme of subunit dissociation. The initial decline in the light-scattering molecular weight curves required one or two apparent binding sites per hemocyanin dimer formed as intermediate dissociation product, with apparent dissociation constants (kD,2) for Ca2+ ions of 0.7 to 7 X 10(-4) M, not very different from the value of 2.5 X 10(-4) M obtained by Makino by equilibrium dialysis for the hemocyanin of the opistobranch, Dolabella auricularia. The binding of Mg2+ ion to S. conspicua and M. muscosa hemocyanins appears to be both weaker than the binding of Ca2+ and more pH dependent, with kD,2 values ranging from the 3 X 10(-4) to 4 X 10(-2) M at pH 8.5 to 9.5. The dissociation the decameric hemocyanin species (sedimentation coefficient ca. 60 S) is also observed in the ultracentrifugation with the initial appearance of 18-20 S dimers, followed by a shift in equilibrium to monomeric species of lower sedimentation rates of 11-12 S as the divalent ion concentration is reduced below 1 X 10(-4) M Ca2+ and Mg2+. The dissociation of dimers to monomers in the second step of the reaction is characterized by one or two binding sites per subunit and a somewhat stronger affinity for divalent ions, indicated by apparent dissociation constants (kD,1) of 0.7 X 10(-4) to 3 X 10(-3) M. Circular dichroism and absorbance measurements at 222 and 346 nm suggest no significant changes in the conformation of the hemocyanin subunits produced by the different stages of subunit dissociation.     

Glutamate substitution in repeat IV alters divalent and monovalent cation permeation in the heart Ca2+ channel.

In voltage-gated ion channels, residues responsible for ion selectivity were identified in the pore-lining SS1-SS2 segments. Negatively charged glutamate residues (E393, E736, E1145, and E1446) found in each of the four repeats of the alpha 1C subunit were identified as the major determinant of selectivity in Ca2+ channels. Neutralization of glutamate residues by glutamine in repeat I (E393Q), repeat III (E1145Q), and repeat IV (E1446Q) decreased the channel affinity for calcium ions 10-fold from the wild-type channel. In contrast, neutralization of glutamate residues in repeat II failed to significantly alter Ca2+ affinity. Likewise, mutation of neighboring residues in E1149K and D1450N did not affect the channel affinity, further supporting the unique role of glutamate residues E1145 in repeat III and E1446 in repeat IV in determining Ca2+ selectivity. Conservative mutations E1145D and E1446D preserved high-affinity Ca2+ binding, which suggests that the interaction between Ca2+ and the pore ligand sites is predominantly electrostatic and involves charge neutralization. Mutational analysis of E1446 showed additionally that polar residues could achieve higher Ca2+ affinity than small hydrophobic residues could. The role of high-affinity calcium binding sites in channel permeation was investigated at the single-channel level. Neutralization of glutamate residue in repeats I, II, and III did not affect single-channel properties measured with 115 mM BaCl2. However, mutation of the high-affinity binding site E1446 was found to significantly affect the single-channel conductance for Ba2+ and Li+, providing strong evidence that E1446 is located in the narrow region of the channel outer mouth. Side-chain substitutions at 1446 in repeat IV were used to probe the nature of divalent cation-ligand interaction and monovalent cation-ligand interaction in the calcium channel pore. Monovalent permeation was found to be inversely proportional to the volume of the side chain at position 1446, with small neutral residues such as alanine and glycine producing higher Li+ currents than the wild-type channel. This suggests that steric hindrance is a major determinant for monovalent cation conductance. Divalent permeation was more complex. Ba2+ single-channel conductance decreased when small neutral residues such as glycine were replaced by bulkier ones such as glutamine. However, negatively charged amino acids produced single-channel conductance higher than predicted from the size of their side chain. Hence, negatively charged residues at position 1446 in repeat IV are required for divalent cation permeation.