Structure of chicken skeletal muscle troponin C at 1.78 Å resolution

Abstract

The structure of chicken skeletal muscle troponin C (TnC) has been refined to an R value of 0.168, using 14 788 reflections, in the resolution range 8.0-1.78 .~,. Our earlier 2 ,A, resolution structure [Satyshur, Rao, Pyzalska, Drendel, Greaser & Sundaralingam (1988).J. Biol. Chem. 263, 1628-1647 served as the starting model. The refined model includes atoms for all protein residues (1-162), 2 Ca 2+ ions, 169 water molecules and one sulfate ion. The high-resolution refinement shows more clearly the details of the protein and water structure. The side chains Glu63, Cysl01, Arg123, Aspl40 and Asp152 adopt two discretely ordered conformations. The long central helix is only slightly curved/bent (7.9 ° ) and all the central helix NH-.-O--C hydrogen bonds are intact. Seven of the nine carbonyl O atoms of the mid segment of this helix, including the D/E linker region, are hydrogen bonded to water molecules which weakens the helix hydrogen bonds. In contrast, in each of the protected upper and lower thirds of the long central helix, only two carbonyl O atoms are hydrogen bonded to water molecules. The hydrogen-bonding patterns displayed by some of the carbonyl O atoms of NT and A helices of the N-terminal domain and the F and H helices of the C-terminal domain, which are on the exposed surface of the protein, are similar. The B helix of the calcium-free site I is kinked, with the local helix axes at either end making an angle of 39 ° , by two inserted water molecules between N--H and O--C groups, breaking the adjacent helix hydrogen bonds. A sul-

Solution secondary structure of calcium‐saturated troponin C monomer determined by multidimensional heteronuclear NMR spectroscopy

“…Chicken skeletal TnC has 162 amino acid residues (for a molecular weight of 18,257) and binds four metal ions in four separate metal ionbinding sites numbered consecutively from the N-terminus of the protein. The structure of half-saturated avian skeletal TnC (with calcium binding sites 111 and IV filled) has been solved using X-ray crystallographic techniques (Herzberg & James, 1988;Satyshur et al, 1988Satyshur et al, , 1994 and reveals two domains (the C-terminal and the N-terminal domains), each with two EF-hand helix-loop-helix calcium binding sites. The C-terminal domain contains the high affinity calcium/magnesium binding sites, which have a Kc, in the range of 2 X IO' M" ; these are referred to as the structural sites because they are always filled with calcium or magnesium in the muscle cell (Potter & Gergely, 1975).…”

“…One residue of particular interest, is the residue Glu 41. This residue was found to have "irregular" non- F22 K23 A24 A25 F26 D27 M28 F29 D30 A3 1 D32 G33 G34 G35 D36 137 S38 T39 K40 E4 1 L42 G43 "44 v45 M46 R47 M48 L49 G50 Q5 1 N52 P53 T54 K55 E56 E57 L58 D59 helical $/$ angles (4 = -96"; $ = -7') in the crystal structure and was shown to form a "kink" in helix B of the crystal structure where the N-domains are free of calcium (Herzberg & James, 1988;Satyshur et al, 1988Satyshur et al, , 1994. The calcium-saturated TnC structure presented here has no indication of $/$ angles for residue Glu 41 other than helix, suggesting that this residue has shifted its $/$ angles to better fit into a helix in the calciumsaturated form.…”

“…The nine helices start and stop at approximately the same residue (Herzberg & James, 1988;Satyshur et al, 1988Satyshur et al, , 1994. The major difference in secondary structure occurs with the residue Glu 41, which is located in helix B.…”

Structural basis for prokaryotic calciummediated regulation by a Streptomyces coelicolor calcium binding protein

“…2). Unlike other helix-loop-helix calcium binding proteins with known structure (Babu et al, 1988;Satyshur et al, 1988;Vijay-Kumar and Cook, 1992;Cook et al, 1993;Tossavainen et al, 2003), all of the non-helical portions of the EF-hand motifs do not contain short antiparallel β-sheets, but instead adopt loop segments. Nevertheless, main chain hydrogen bonds do occur between residues Ile25 and Ile76, which occupy the eighth position of each N-terminal calcium binding loop.…”

An NMR and spin label study of the effects of binding calcium and troponin I inhibitory peptide to cardiac troponin C

“…TnC is a member of the helix-loop-helix or EF-hand family of Ca2+ binding proteins (Kretsinger, 1980). Crystallographic structures of sTnC reveal a dumbbell-shaped molecule with N-and C-terminal globular domains joined by a helical linker (Herzberg & James, 1985;Satyshur et al, 1988). Each domain contains two EF-hand Ca2+ binding sites.…”