Parvalbumin, calcium binding sites

Parvalbumin is a muscle protein with a single polypeptide chain of 109 amino acids. Its function is uncertain, but calcium binding to this protein probably plays a role in muscle relaxation. The helix-loop-helix motif appears three times in this structure, in two of the cases there is a calcium-binding site. Figure 2.13 shows this motif which is called an EF hand because the fifth and sixth helices from the amino terminus in the structure of parvalbumin, which were labeled E and F, are the parts of the structure that were originally used to illustrate calcium binding by this motif. Despite this trivial origin, the name has remained in the literature. 

Parvalbumin, 46 443, 445 calcium binding sites, 42 113-114 p backbonding, osmium complexes, 37 316, 320... 

Using the Forster equation the distance between the two calcium-binding sites in parvalbumin (Fig. 6-7) has been estimated by energy transfer from Eu(III) in one site to Tb(III) in the other165 to within 10-15% of the distance of 1.18 nm based on X-ray crystallography. 

Lanthanides have been used as substitutes for calcium in calcium binding proteins since the early days in NMR [155,156]. Remarkable information was obtained on systems such as Yb(III)-substituted parvalbumin (which contains a typical calcium binding site called EF-hand [157,158]) from ID spectroscopy alone [159-164], later complemented by 2D spectroscopy [165]. More recently, pseudocontact shifts and longitudinal proton relaxation times have been used to... 

Calmodulin has a MW % 16,700 and its primary sequence is homologous with that of parvalbumins and skeletal muscle TnC. Its amino acid sequence can be divided into four internally homologous domains, each with a potential calcium binding site(16). Most studies have indeed indicated that calmodulin can bind 4 mol of Ca " per mol of protein but there is some disagreement as regards the relative number of high and low affinity sites. 

This is the method applied in our study of the sodium interaction with half-decalcified parvalbumin. The alkali ion binds to the calcium-binding site of this muscular protein vacated by the Ca" " ion. The rate constants for the equilibrium (5j ... 

MOLECULAR GRAPHICS IN THE STUDY OF THE CALCIUM-BINDING SITES OF CARP PARVALBUMIN AND OTHER PROTEINS... 

Lockhart, J. C. cuad H. Grey - Molecular graphics in the study of the calcium-binding sites of carp parvalbumin and other proteins 113... 

Nonrepetitive but well-defined structures of this type form many important features of enzyme active sites. In some cases, a particular arrangement of coil structure providing a specific type of functional site recurs in several functionally related proteins. The peptide loop that binds iron-sulfur clusters in both ferredoxin and high potential iron protein is one example. Another is the central loop portion of the E—F hand structure that binds a calcium ion in several calcium-binding proteins, including calmodulin, carp parvalbumin, troponin C, and the intestinal calcium-binding protein. This loop, shown in Figure 6.26, connects two short a-helices. The calcium ion nestles into the pocket formed by this structure. 

Fig. 13. The binding sites of calcium in (a) parvalbumin (41a), (b) annexin (41) and (c) calmodulin (42). The drawings show two bidentate carboxylates coordinated to Ca2 in the EF-hand site of parvalbumin, and one bidentate carboxylate coordinated to Ca2 in annexin and calmodulin. All the donor atoms coordinated to the calciums are oxygen donor atoms from carboxylates of asp = aspartate, or glu = glutamate, or else peptide carbonyl oxygens from gly = glycine or met = methionine. Redrawn after Refs. (41-42).

A protein of similar molecular weight to that of rat oncomodulin, rat and rabbit parvalbumins, S100, and the vitamin D-dependent calcium-binding proteins has been isolated from chicken gizzard smooth muscle. In this case, however, the fluorescence emission from the four tyrosine residues is quenched by Ca2+ binding.(160) The decrease in fluorescence intensity was used to suggest that there are two different classes of Ca2+binding sites. 

Parvalbumins, which are also found in other vertebrates, are high-affinity Ca2+-buffers." Additional calcium buffers with EF-hand structures are the vitamin D-induced calbindins. One 9-kDa calbindin is found in mammalian intestinal tissue and in skin. It has two helix-loop-helix Ca2+-binding sites of differing affinity101102 that presumably function in the absorption of calcium. A 28-kDa vitamin D-dependent protein from chicken intestine contains six similar Ca2 +-binding loops.97 103... 

The calcium sites in troponin C have been studied by X-ray absorption near edge structure (XANES).244 In all four cases, Ca2+ appears to be coordinated to carboxylate and carbonyl groups, and no structural differences could be found between the two classes of sites. Binding of Mg2+ causes a distortion of the geometry of the calcium site. Thus, the reduced affinity for Ca2+ of the Ca2+-Mg2+ sites in the presence of Mg2+ may not simply be due to competition with Mg2+, but due to some conformational change induced at these sites by Mg2+. The similarity of all four Ca2+ sites means that local bonding effects do not explain the inability of Mg2+ to bind to the calcium-specific sites I and II. The XANES of parvalbumin differs from that of troponin C. 

These are soluble, sarcoplasmic calcium-binding proteins found in vertebrates. Invertebrates contain different sarcoplasmic calcium-binding proteins.247 Parvalbumins are low molecular weight (10 000-12 000), acidic proteins which contain two binding sites for Ca2+. The structure of parvalbumin has been determined, and details of the calcium sites are given in Table 6. XANES studies indicate that the calcium sites are similar to those in calmodulin, but different from troponin C.244,248 The two parvalbumin sites have different coordination numbers, six and eight.249... 

Redesign of Ca fMg Specificity. Proteins in the calmodulin superfamily (including troponin C, parvalbumin (PV) and oncomodulin (OM)) share similar overall structure and yet have different selectivity for Ca and Mg see Calcium-binding Proteins, Cation-activated Enzymes). For example, PV and OM have four helix-tum-helix domains, two of which contain mixed Ca /Mg sites and two Ca -specific sites. The mixed Ca /Mg sites have been converted to Ca -specific sites by replacing amino acids with the corresponding residues in the Ca -specific site. " ... 

Contrary to the previous case, parvalbumin binds only two Ca ions. Compared to the technical difficulties encountered at the K-absorption edge of calcium due to increased absorption of 3 A wavelengths, the quantitative replacement of Ca by terbium ions (Lj-edge at 1.648 A) offers an ideal way to obtain structural information about the ion binding sites of parvalbumin by resonance scattering experiments. In fact, Miake-Lye, Doniach and Hodgson were able to determine for the first time the distance between the center of mass of the parvalbumin and its two terbium ion binding sites in solution. 

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