Fragment complementation of calbindin D28k

Calbindin D28k is a highly conserved Ca2+-binding protein abundant in brain and sensory neurons. The 261-residue protein contains six EF-hands packed into one globular domain. In this study, we have reconstituted calbindin D28k from two fragments containing three EF-hands each (residues 1-132 and 133-261, respectively), and from other combinations of small and large fragments. Complex formation is studied by ion-exchange and size-exclusion chromatography, electrophoresis, surface plasmon resonance, as well as circular dichroism (CD), fluorescence, and NMR spectroscopy. Similar chromatographic behavior to the native protein is observed for reconstituted complexes formed by mixing different sets of complementary fragments, produced by introducing a cut between EF-hands 1, 2, 3, or 4. The C-terminal half (residues 133-261) appears to have a lower intrinsic stability compared to the N-terminal half (residues 1-132). In the presence of Ca2+, NMR spectroscopy reveals a high degree of structural similarity between the intact protein and the protein reconstituted from the 1-132 and 133-261 fragments. The affinity between these two fragments is 2 x 10(7) M(-1), with association and dissociation rate constants of 2.7 x 10(4) M(-1) s(-1) and 1.4 x 10(-3) s(-1), respectively. The complex formed in the presence of Ca2+ is remarkably stable towards unfolding by urea and heat. Both the complex and intact protein display cold and heat denaturation, although residual alpha-helical structure is seen in the urea denatured state at high temperature. In the absence of Ca2+, the fragments do not recombine to yield a complex resembling the intact apo protein. Thus, calbindin D28k is an example of a protein that can only be reconstituted in the presence of bound ligand. The alpha-helical CD signal is increased by 26% after addition of Ca2+ to each half of the protein. This suggests that Ca2+-induced folding of the fragments is important for successful reconstitution of calbindin D28k.     

Domain organization of calbindin D28k as determined from the association of six synthetic EF-hand fragments.

Calbindin D28k is an intracellular Ca(2+)-binding protein containing six subdomains of EF-hand type. The number and identity of the globular domains within this protein have been elucidated using six synthetic peptide fragments, each corresponding to one EF-hand subdomain. All six peptides were mixed in equimolar amounts in the presence of 10 mM Ca2+ to allow for the reconstitution of domains. The mixture was compared to native calbindin D28k and to the sum of the properties of the individual peptides using circular dichroism (CD), fluorescence, and 1H NMR spectroscopy, as well as gel filtration and ion-exchange chromatography. It was anticipated that if the peptides associate to form native-like domains, the properties would be similar to those of the intact protein, whereas if they did not interact, they would be the same as the properties of the isolated peptides. The results show that the peptides in the mixture interact with one another. For example, the CD and fluorescence spectra for the mixture are very similar to those of the intact calbindin D28k, suggesting that the mixed EF-hand fragments associate to form a native-like structure. To determine the number of domains and the subdomain composition of each domain in calbindin D28k, a variety of peptide combinations containing two to five EF-hand fragments were studied. The spectral and chromatographic properties of all the mixtures containing less than six peptides were closer to the sum of the properties of the relevant individual peptides than to the mixture of the six peptides. The results strongly suggest that all six EF-hands are packed into one globular domain. The association of the peptide fragments is observed to drive the folding of the individual subdomains. For example, one of the fragments, EF2, which is largely unstructured in isolation even in the presence of high concentrations of Ca2+, is considerably more structured in the presence of the other peptides, as judged by CD difference spectroscopy. The CD data also suggest that the packing between the individual subdomains is specific.     

Characterization of calretinin I-II as an EF-hand, Ca2+, H+-sensing domain

Calretinin, a neuronal protein with well-defined calcium-binding properties, has a poorly defined function. The pH dependent properties of calretinin (CR), the N-terminal (CR I-II), and C-terminal (CR III-VI) domains were investigated. A drop in pH within the intracellular range (from pH 7.5 to pH 6.5) leads to an increased hydrophobicity of calcium-bound CR and its domains as reported by fluorescence spectroscopy with the hydrophobic probe 2-(p-toluidino)-6-naphthalenesulfonic acid (TNS). The TNS data for the N- and C-terminal domains of CR are additive, providing further support for their independence within the full-length protein. Our work concentrated on CR I-II, which was found to have hydrophobic properties similar to calmodulin at lower pH. The elution of CR I-II from a phenyl-Sepharose column was consistent with the TNS data. The pH-dependent structural changes were further localized to residues 13-28 and 44-51 using nuclear magnetic resonance spectroscopy chemical shift analysis, and there appear to be no large changes in secondary structure. Protonation of His 12 and/or His 27 side chains, coupled with calcium chelation, appears to lead to the organization of a hydrophobic pocket in the N-terminal domain. CR may sense and respond to calcium, proton, and other signals, contributing to conflicting data on the proteins role as a calcium sensor or calcium buffer.     

Characterization of the N-terminal half-saturated state of calbindin D9k: NMR studies of the N56A mutant.

Calbindin D9k is a small EF-hand protein that binds two calcium ions with positive cooperativity. The molecular basis of cooperativity for the binding pathway where the first ion binds in the N-terminal site (1) is investigated by NMR experiments on the half-saturated state of the N56A mutant, which exhibits sequential yet cooperative binding (Linse S, Chazin WJ, 1995, Protein Sci 4:1038-1044). Analysis of calcium-induced changes in chemical shifts, amide proton exchange rates, and NOEs indicates that ion binding to the N-terminal binding loop causes significant changes in conformation and/or dynamics throughout the protein. In particular, all three parameters indicate that the hydrophobic core undergoes a change in packing to a conformation very similar to the calcium-loaded state. These results are similar to those observed for the (Cd2+)1 state of the wild-type protein, a model for the complementary half-saturated state with an ion bound in the C-terminal site (II). Thus, with respect to cooperativity in either of the binding pathways, binding of the first ion drives the conformation and dynamics of the protein far toward the (Ca2+)2 state, thereby facilitating binding of the second ion. Comparison with the half-saturated state of the analogous E65Q mutant confirms that mutation of this critical bidentate calcium ligand at position 12 of the consensus EF-hand binding loop causes very significant structural perturbations. This result has important implications regarding numerous studies that have utilized mutation of this critical residue for site deactivation.     

Calbindin‐D28k and calretinin in chicken inner retina during postnatal development and neuroplasticity by dim red light

Members of the family of calcium binding proteins (CBPs) are involved in the buffering of calcium (Ca2+) by regulating how Ca2+ can operate within synapses or more globally in the entire cytoplasm and they are present in a particular arrangement in all types of retinal neurons. Calbindin D28k and calretinin belong to the family of CBPs and they are mainly co‐expressed with other CBPs. Calbindin D28k is expressed in doubles cones, bipolar cells and in a subpopulation of amacrine and ganglion neurons. Calretinin is present in horizontal cells as well as in a subpopulation of amacrine and ganglion neurons. Both proteins fill the soma at the inner nuclear layer and the neuronal projections at the inner plexiform layer. Moreover, calbindin D28k and calretinin have been associated with neuronal plasticity in the central nervous system. During pre and early postnatal visual development, the visual system shows high responsiveness to environmental influences. In this work we observed modifications in the pattern of stratification of calbindin immunoreactive neurons, as well as in the total amount of calbindin through the early postnatal development. In order to test whether or not calbindin is involved in retinal plasticity we analyzed phosphorylated p38 MAPK expression, which showed a decrease in p‐p38 MAPK, concomitant to the observed decrease of calbindin D28k. Results showed in this study suggest that calbindin is a molecule related with neuroplasticity, and we suggest that calbindin D28k has significant roles in neuroplastic changes in the retina, when retinas are stimulated with different light conditions. © 2013 Wiley Periodicals, Inc. Develop Neurobiol 73: 530–542, 2013     

Deamidation and disulfide bridge formation in human calbindin D28k with effects on calcium binding

Calbindin D(28k) (calbindin) is a cytoplasmic protein expressed in the central nervous system, which is implied in Ca(2+) homeostasis and enzyme regulation. A combination of biochemical methods and mass spectrometry has been used to identify post-translational modifications of human calbindin. The protein was studied at 37 degrees C or 50 degrees C in the presence or absence of Ca(2+). One deamidation site was identified at position 203 (Asn) under all conditions. Kinetic experiments show that deamidation of Asn 203 occurs at a rate of 0.023 h(-1) at 50 degrees C for Ca(2+)-free calbindin. Deamidation is slower for the Ca(2+)-saturated protein. The deamidation process leads to two Asp iso-forms, regular Asp and iso-Asp. The form with regular Asp 203 binds four Ca(2+) ions with high affinity and positive cooperativity, i.e., in a very similar manner to non-deamidated protein. The form with beta-aspartic acid (or iso-Asp 203) has reduced affinity for two or three sites leading to sequential Ca(2+) binding, i.e., the Ca(2+)-binding properties are significantly perturbed. The status of the cysteine residues was also assessed. Under nonreducing conditions, cysteines 94 and 100 were found both in reduced and oxidized form, in the latter case in an intramolecular disulfide bond. In contrast, cysteines 187, 219, and 257 were not involved in any disulfide bonds. Both the reduced and oxidized forms of the protein bind four Ca(2+) ions with high affinity in a parallel manner and with positive cooperativity.     

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.     

A calbindin D9k mutant containing a novel structural extension: 1H nuclear magnetic resonance studies.

Calbindin D9k is a small, well-studied calcium-binding protein consisting of two helix-loop-helix motifs called EF-hands. The P43MG2 mutant is one of a series of mutants designed to sequentially lengthen the largely unstructured tether region between the two EF-hands (F36-S44). A lower calcium affinity for P43MG was expected on the basis of simple entropic arguments. However, this is not the case and P43MG (-97 kJ.mol-1) has a stronger calcium affinity than P43M (-93 kJ.mol-1), P43G (-95 kJ.mol-1) and even wild-type protein (-96 kJ.mol-1). An NMR study was initiated to probe the structural basis for these calcium-binding results. The 1H NMR assignments and 3JHNH alpha values of the calcium-free and calcium-bound form of P43MG calbindin D9k mutant are compared with those of P43G. These comparisons reveal that little structure is formed in the tether regions of P43MG(apo), P43G(apo) and P43G(Ca) but a helical turn (S38-K41) appears to stabilize this part of the protein structure for P43MG(Ca). Several characteristic NOEs obtained from 2D and 3D NMR experiments support this novel helix. A similar, short helix exists in the crystal structure of calcium-bound wild-type calbindin D9k-but this is the first observation in solution for wild-type calbindin D9k or any of its mutants.     

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.     

The effect of different maternal deprivation paradigms on the expression of hippocampal glucocorticoid receptors, calretinin and calbindin-D28k in male and female adolescent rats

Maternal deprivation (MD) is a well-established protocol used to investigate neurobiological changes that are associated with the etiology of and vulnerability to stress-related diseases in animal models. The resulting psychophysiological effects, the timing and duration of these adverse stimuli, and the method by which they exert their effects on the animals remain unclear. This study characterized differences in the hippocampal expression of glucocorticoid receptors (GRs) and the calcium-binding proteins calretinin (CALR) and calbindin-D28k (CALB) in male and female rats that underwent different MD paradigms during the stress hyporesponsive period (SHRP). Both GRs and the two calcium-binding proteins were much more abundant in females than in males. MD paradigms had a significant effect on CALR and CALB expression in both males and females but affected GR levels only in males. Additionally, expression of the two calcium-binding proteins in the hippocampus responded differently to MD-induced stress, especially in females. Taken together, these results indicate that females are able to modulate their response to stress better than males.     

Estrogen responsiveness of renal calbindin-D28k gene expression in rat kidney

In women, calcium excretion in the urine rises after menopause and falls with estrogen replacement therapy. The amount of calcium lost in the urine following estrogen therapy is less than should occur based on changes in serum calcium and the amount of calcium filtered by the kidney. This suggests there may be a direct effect of estrogen therapy to increase renal calcium reabsorption. Calbindin D_28k is a putative calcium ferry protein located in the distal renal tubules which has been shown to increase transcellular calcium transport. We proposed that estrogen loss after menopause may diminish gene expression of renal calbindin D_28k and subsequently diminish renal calcium reabsorption. We used the ovariectomized rat model of estrogen deficiency to investigate changes at the messenger RNA level of calbindin D_28k in ovariectomized rats (OVX), sham ovariectomized rats (S-OVX), and estrogen treated ovariectomized rats (E-OVX). We have demonstrated that ovariectomy in rats diminishes the gene expression of renal calbindin D_28k. The mRNA levels were approximately three times lower in OVX rats than S-OVX rats. Administration of 17β estradiol to OVX rats produced a significant increase in mRNA level to greater than the S-OVX rats by 4 h. Measurement of serum 1,25 dihydroxyvitamin D₃ showed lower level in OVX rats than S-OVX rats but no significant change in E-OVX animals. In conclusion, our results indicate that estrogen increases renal calbindin D_28k mRNA levels, by a mechanism independent of changes in 1,25 dihydroxyvitamin D₃. This may result in increased expression of calbindin D_28k protein which may have a role in reducing renal calcium excretion. J. Cell. Biochem. 65:340–348. © 1997 Wiley-Liss, Inc.     

Calretinin and calbindin in the retina of the developing chick

Summary Calretinin and calbindin-D28k are two calcium-binding proteins that are present in largely different sets of nerve cells in the central nervous system. Their appearance during development of the chick retina was studied by immunohistochemistry and Western blots. The patterns are mature one day before hatching. Each cell type acquires its characteristic calcium-binding protein several days after its differentiation has started, but in most cases before morphological maturation is complete. There is also an early phase of calbindin immunoreactivity in many immature amacrine cells, and of calretinin immunoreactivity in the presumptive photoreceptor layer, suggesting that these proteins may have distinct functions in differentiating cells.Abbreviations CR+ Immunoreactive for calretinin only - CB+ immunoreactive for calbindin only - CR+CB+ immunoreactive for both antisera - IPL inner plexiform layer - OPL outer plexiform layer     

Apoptosis- and endoplasmic reticulum stress-related genes were regulated by estrogen and progesterone in the uteri of calbindin-D(9k) and -D(28k) knockout mice

Calcium (Ca(2+)) is an important regulator of apoptotic signaling. Calbindin-D(9k) (CaBP-9k) and -D(28k) (CaBP-28k) have a high affinity for Ca(2+) ions. Uterine calbindins appear to be involved in the regulation of myometrial activity by intracellular Ca(2+). In addition, uterine calbindins are expressed in the mouse endometrium and are regulated by steroid hormones during implantation and development. The aim of the present study was to evaluate the regulation of apoptosis in the uteri of CaBP-9k, CaBP-28k, and CaBP-9k/28k knockout (KO) mice. Our findings indicated that Bax protein was enhanced in the uteri of CaBP-28k and CaBP-9k/28k KO mice compared to wild-type (WT) and CaBP-9k KO mice, but no difference was observed in Bcl-2 protein expression. The expressions of caspase 3, 6, and 7 proteins were higher in both CaBP-28k and CaBP-9k/28k KO mice than in WT and CaBP-9k KO mice. These results suggest that the absence of CaBP-28k increases apoptotic signaling. We also investigated the expression of endoplasmic reticulum (ER) stress genes by Western blot analysis in calbindin KO mice. C/EBP homologous protein and immunoglobulin heavy chain-binding protein protein levels were elevated in CaBP-28k KO mice compared to WT mice. When immature mice were treated with 17β-estradiol (E2) or progesterone (P4) for 3 days, we found that the expressions of Bax and caspase 3 protein were increased by E2 treatment in WT and CaBP-9k KO mice, and by P4 treatment in CaBP-28k KO mice. These results indicate that CaBP-28k blocks the up-regulation of apoptosis-related genes and ER stress genes, implying that CaBP-28k may decrease the expression of genes involved in apoptosis and ER stress in murine uterine tissue.     

Calbindin D28k is essentially located in the colonic part of the toad intestine

Summary— The distribution of calbindin D28k in the digestive system and the urinary bladder of the toad was investigated using immunohistochemistry and Western blotting. By analogy with mammals and birds, the protein was expected to be located preferentially in the duodenal part of the intestine. Interestingly, absorptive cells of the duodenum were totally devoid of calbindin D28k while the colon contained high amounts of the calcium-binding protein. This reversed polarity of calbindin D28k content in the toad intestine should obviously correspond to a different scheme of calcium absorption regulation between amphibians and higher vertebrates. Calbindin D28k containing neuroendocrine-like cells were found scattered in the proximal parts of the gut with a similar distribution to what has been described in rat and chick intestine. The oesophagus, the stomach, and the intrinsic nervous sytem of the intestine were negative. No significant amounts of the proteins were found in the urinary bladder, which is known to be a site of Ca²⁺ active transport.     

Impact of subchronic exposure to triclosan and/or fluoride on estrogenic activity in immature female rats: The expression pattern of calbindin‐D9k and estrogen receptor α genes

This study explored the influence of triclosan (TCS) in the absence and presence of sodium fluoride (NaF) on estrogenic activity and thyroid function of adolescent female rats. The results indicated that the individual exposure to TCS evoked a significant decline in T₃ and T₄ but the levels of estradiol, FSH, and LH were significantly elevated beside marked up regulation of calbindin‐D9k and estrogen α mRNA expression. On the other hand, the single exposure to NaF causes insignificant changes in thyroid hormones, but evoked a trend toward an increase in both estradiol and LH levels. No significant differences in the TSH level were recorded among the experimental groups. The joint exposure to TCS and NaF induced a significant improvement in thyroid and reproductive hormone levels. Overall, these findings revealed that exposure to TCS resulted in significant endocrine and reproductive alterations in immature female rats, while TCS + NaF coexposure resulted in lessening most effects.