Troponin calcium sites

Magnesium is also of interest as a replacement for Ca(ll) in calcium-requiring enzymes. In some of these, the replacement is simple (Lewinski and Lebioda, 1986), and in others it cannot occur. NMR studies show that magnesium can bind in the calcium sites of troponin C (Tsuda et al., 1990). The structure of turkey skeletal muscle troponin C has recently been reported (Herzberg and James, 1985). In one domain the replacement of Ca(II) by Mg(II) causes a conformational change, but in the other domain it does not. 

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... 

Schematic diagram of a cardiac muscle sarcomere, with sites of action of several drugs that alter contractility (numbered structures). Site 1 is Na+/K+ ATPase, the sodium pump. Site 2 is the sodium/calcium exchanger. Site 3 is the voltage-gated calcium channel. Site 4 is a calcium transporter that pumps calcium into the sarcoplasmic reticulum (SR). Site 5 is a calcium channel in the membrane of the SR that is triggered to release stored calcium by activator calcium. Site 6 is the actin-troponin-tropomyosin complex at which activator calcium brings about the contractile interaction of actin and myosin.

In the light of the available crystallographic studies, the amino acid sequences and NMR studies, certain general points have been noted for parvalbumin, Wasserman protein, calmodulin, troponin C and SIOO. (1) Each calcium site is formed from residues in a hand which includes a /3-strand. The sites contain backbone carbonyl and side-chain carboxylate. (2) Each binding site is linked to two helices. (3) Each -strand backs on to another strand to form a Ca-/3-sheet-Ca unit, which then involves four helices. (4) The helices interact with each other through largely hydrophobic surfaces. (5) The connections between remote ends of helices are relatively mobile strands, and differ from protein to protein. (6) The four-hand proteins, the calmodulins and troponins, are simileir to the sum of their two-handed fragments. 

Because of our interest in utilizing Cd NMR spectroscopy as a probe for calcium sites in biological systems (42,43,49,51), we have been interested in the isotropic Cd chemical shifts in these compounds. In the systems studied (Concanavalin A (42), Parvalbumin (43), Troponin C (50),. Calmodulin (49), and Insulin (51)), replacing calcium with cadmium places cadmium in an environment in which all of the atoms in the first coordination sphere are oxygens. The cadmium is assumed to be six coordinate. The isotropic chemical shifts for these compounds (82) fall into a characteristic range of chemical shifts from -85 to -130 ppm with respect to 0.1 M Cd(C104)2 To date, there are no model compounds that can be studied in aqueous solutions which have isotropic shifts within this range. 

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. 

Tropomyosin is thought to lie in the groove formed between the associated actin strands. The sites at which the myosin crossbridges attach are affected by the relationship between tropomyosin and the actin strands. The role of tropomyosin in smooth muscle is completely undefined while in striated muscle it is clearly involved in the activation of contraction. The difference is made clear by the absence from smooth muscle of the protein, troponin, which in striated muscle provides the binding site for the triggering calcium. 

Site of calcium binding Troponin Calmodulin Calmodulin... 

Function of calcium Reposition troponin/tropomyosin to uncover myosin binding sites on actin Phosphorylate and activate myosin to bind with actin Phosphorylate and activate myosin to bind with actin... 

In skeletal muscle, calcium binds to troponin and causes the repositioning of tropomyosin. As a result, the myosin-binding sites on the actin become uncovered and crossbridge cycling takes place. Although an increase in cytosolic calcium is also needed in smooth muscle, its role in the mechanism of contraction is very different. Three major steps are involved in smooth muscle contraction ... 

Johnson, J.D., Collins, J.H., and Potter, J.D. (1978) Dansylaziridine labeled troponin C. A fluorescent probe of calcium ion binding to the calcium ion-specific regulatory sites./. Biol. Chem. 253, 6451. 

Calcium binds to troponin C on tropomyosin, causing a conformational change that exposes myosin-binding sites on actin. 

P. Kanellis, J. Yang, H. C. Cheung, and R. E. Lenkinski, Synthetic peptide analogs of skeletal troponin C Fluorescence studies of analogs of the low-affinity calcium-binding site II, Arch. Biochem. Biophys. 220, 530-540 (1983). 

N. A. Malik, G. M. Anatharamaiah, A. Gawish, and H. C. Cheung, Structural and biological studies on synthetic peptide analogues of a low-affinity calcium-binding site of skeletal troponin C, Biochim. Biophys. Acta 911, 221-230 (1987). 

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