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3.13: Paravalbumin and Calbindins \(D_{9K}\) and \(D_{28K}\)

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    60281
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    A few intracellular Ca2+-binding proteins have been discovered that by sequence homology clearly belong to the CaM-TnC family with Ca2+ sites of the "EF-hand"-type, but that do not appear to exert a direct regulatory function. Parvalbumins (Mr ≈ 12 kDa), calbindin D9K (Mr ≈ 8.7 kDa) and calbindin D28K (Mr ≈ 28 kDa) belong to this group. Parvalbumin(s) exist in two main types, \(\alpha\) and \(\beta\), found in large quantities in the white muscle of fish, amphibia, and reptiles, but also in different mammalian tissues,116,117 including neurons of the central and peripheral nervous system. The molecule has two fairly strong Ca2+- binding sites (see Table 3.2). The x-ray structure of carp parvalbumin was solved in 1973 by Kretsinger et al.,118 and for a decade provided the basis for all discussions on intracellular Ca2+-binding proteins. The concept of the canonical "EF-hand" Ca2+-binding site originated from the parvalbumin work, and the name "EF" derives from the labeling of the two helices that flank the second of the two Ca2+ sites in parvalbumin, as shown in Figure 3.23.

    clipboard_e3be2f1cdefe7bf1b2d97e2c8a8cc2abc
    Figure 3.23 - Structure of the Ca2+-binding sites of carp parvalbumin. The Ca2+ ions are depicted as regular octahedra making six ligand contacts with oxygen atoms at each vertex, labeled x, y, z, -x, -y, -z. The helix-loop-helix structure that forms a Ca2+-binding site can be regarded as a hand with the forefinger representing one helix (e.g., the E-helix) in the plane of the figure, the thumb oriented perpendicular to the plane representing the second helix (the F-helix), and the remaining fingers make up the Ca2+-binding loop. After Kretsinger and Barry.118

    If the first Ca2+ ligand in the approximately octahedral coordination sphere is given number 1 (or "x") the others come in the order 3( "y"), 5("z"), 7("-y"), 9("-x"), and 12("-z"). In the second site of parvalbumin, "-x" is actually a H2O molecule, but in the first site it is the carboxylate of a Glu. Studies118 of putative Ca2+-binding sites in other proteins with known primary sequences led to the generalized EF-hand structure—including residues in the flanking \(\alpha\)-helices—shown in Figure 3.24.

    clipboard_e6d17156211eeb6084fc33e220ed5471c
    Figure 3.24 - One consensus EF-hand sequence including residues in the flanking \(\alpha\)-helices; x, y, z, -x, -y, - z denote positions in the octahedral Ca2+ coordination sphere. E—glutamic acid residue, G—glycine residues, I—isoleucine residue, n—nonpolar residue, ♦—a residue with a nonaromatic oxygen-containing side chain (i.e., Glu, Gin, Asp, Asn, Ser, or Thr), and •—nonspecific residue.

    This sequence, with minor modifications, has been widely used in searching for "EF-hands" in libraries of amino-acid (or DNA) sequences of new proteins with unknown properties. In this way, calbindin D28k a protein with unknown function, initially discovered in chicken intestine, but later found also in brain, testes, and other tissue, has been shown to have four EF-hand sites.119

    Recently two structures of carp parvalbumin, both with a resolution of 1.6 Å, were published.120 One of these structures is the native calcium-loaded form of the protein; the second is the structure of parvalbumin in which Ca2+ has been replaced by Cd2+. No significant differences are observed upon replacement of calcium by cadmium. 113Cd has a nuclear spin of I = \(\frac{1}{2}\), making it much more amenable to NMR studies than the quadrupolar 43Ca (I = \(\frac{7}{2}\)), This study supports the use of 113Cd NMR as a tool for the study of calcium-binding proteins.121

    The function of parvalbumin has long been assumed to be that of buffering Ca2+ in muscle cells, i.e., taking up Ca2+ ions released from Ca2+-troponin complexes, thereby ensuring that the cytoplasmic levels of free Ca2+ are always kept very low, even during short bursts of muscle activity,122 The widespread occurrence of parvalbumin in non-muscle tissue indicates that it probably has other roles as well.

    Calbindin D9k (Mr ≈ 8.7 kDa) is another intracellular Ca2+-binding protein with unknown function. It was briefly mentioned in connection with Ca2+ uptake and transport in the intestine and placenta (Section IV.A). Like the avian calbindin D28k the D9k calbindin has been observed in many types of tissue. The homology between the D9k and D28k calbindins is much less than the name suggests; both their syntheses are, however, regulated by vitamin D. The x-ray structure of bovine calbindin D9k has been determined123 and refined to a resolution of 2.3 Å, and a three-dimensional solution structure of porcine calbindin D9k is also available.124 The average solution structure calculated from NMR data is shown in Figure 3.25 (See color plate section, page C-10.)

    The protein has four main \(\alpha\)-helices and two Ca2+-binding loops (I and II). The interior of the molecule shows a loose clustering of several hydrophobic side chains; in particular, three phenylalanine rings come very close in space. The Ca2+-binding loops constitute the least-mobile parts of the molecule. The crystallographic temperature factors have pronounced minima in these regions, with the lowest overall B-factor observed in loop II. Both Ca2+ ions are roughly octahedrally coordinated with protein oxygen atoms. There are some striking differences between the two sites, however. Whereas the C-terminal site (II) has a general structure very similar to the archetypal "EF-hand," as observed in CaM, sTnC, and parvalbumin, the N-terminal site (I) has an extra amino-acid residue inserted between vertices x and y, and z and -y (see Figure 3.24). As a consequence, the peptide fold in site I is different from that in site II. Three carboxylate groups are ligands in site II, but in site I there is only one.

    Despite this marked difference in charge and peptide fold, the Ca2+ affinity of both Ca2+ sites is remarkably similar, as has been shown in a study in which site-directed mutagenesis was combined with different biophysical measurements.37 Cooperative Ca2+ binding in the native calbindin D9k (the "wild type") was first demonstrated at low ionic strength by means of the values of the two stoichiometric Ca2+-binding constants, K1 and K2, which could be measured with good accuracy (K1 = 4.4 x 108 M-1 and K2 = 7.4 x 108 M-1). The effects of amino-acid substitutions in Ca2+ site I were primarily localized to this site, with virtually no effects on the structure or other biophysical properties pertinent to site II. The appearance of sequential Ca2+ binding in some of the calbindin mutants did allow the identification of 1H NMR resonances that respond primarily to binding of Ca2+ to either one of the sites. This result in tum permitted an estimate of the ratio between the site-binding constants (KA and KB) in the wild-type protein and in one of the mutant proteins (Tyr-13 → Phe). In this way the reseachers125 could assess, to within narrow limits, the free energy of interaction, \(\Delta \Delta\)G, between the two Ca2+ sites as 7.7 kJ/mol at low ionic strength and 4.6 kJ/mol in the presence of 0.15 M KCl. How this site-site interaction is transmitted on a molecular level is still unknown.

    Through a combination of site-specific mutations and biophysical measurements, it has recently been demonstrated that carboxylate groups at the surface of the protein, but not directly ligated to the bound Ca2+ ions, have a profound effect on the Ca2+ affinity.126 Neutralization of the surface charges reduces affinity and increases the stability of the protein toward unfolding by urea.127

    A surprising discovery about the structure of bovine calbindin D9k in solution has also been made recently.128 Detailed analysis of the 2D 1H NMR spectrum of wild-type calbindin has revealed that it exists as a 3:1 equilibrium mixture of two forms, corresponding to a trans and cis conformation around the Gly-42-Pro-43 peptide bond. The global fold appears essentially the same in the two forms, and structural differences are primarily located in the inter-domain loop in which Pro-43 is located.


    3.13: Paravalbumin and Calbindins \(D_{9K}\) and \(D_{28K}\) is shared under a CC BY-NC-SA 4.0 license and was authored, remixed, and/or curated by LibreTexts.