Solution structure of human cardiac troponin C in complex with the green tea polyphenol, (-)-epigallocatechin 3-gallate

Abstract

Heart muscle contraction is regulated by Ca(2+) binding to the thin filament protein troponin C. In cardiovascular disease, the myofilament response to Ca(2+) is often altered. Compounds that rectify this perturbation are of considerable interest as therapeutics. Plant flavonoids have been found to provide protection against a variety of human illnesses such as cancer, infection, and heart disease. (-)-Epigallocatechin gallate (EGCg), the prevalent flavonoid in green tea, modulates force generation in isolated guinea pig hearts (Hotta, Y., Huang, L., Muto, T., Yajima, M., Miyazeki, K., Ishikawa, N., Fukuzawa, Y., Wakida, Y., Tushima, H., Ando, H., and Nonogaki, T. (2006) Eur. J. Pharmacol. 552, 123-130) and in skinned cardiac muscle fibers (Liou, Y. M., Kuo, S. C., and Hsieh, S. R. (2008) Pflugers Arch. 456, 787-800; and Tadano, N., Yumoto, F., Tanokura, M., Ohtsuki, I., and Morimoto, S. (2005) Biophys. J. 88, 314a). In this study we describe the solution structure of the Ca(2+)-saturated C-terminal domain of troponin C in complex with EGCg. Moreover, we show that EGCg forms a ternary complex with the C-terminal domain of troponin C and the anchoring region of troponin I. The structural evidence indicates that the binding site of EGCg on the C-terminal domain of troponin C is in the hydrophobic pocket in the absence of troponin I, akin to EMD 57033. Based on chemical shift mapping, the binding of EGCg to the C-terminal domain of troponin C in the presence of troponin I may be to a new site formed by the troponin C.troponin I complex. This interaction of EGCg with the C-terminal domain of troponin C.troponin I complex has not been shown with other cardiotonic molecules and illustrates the potential mechanism by which EGCg modulates heart contraction.

FIGURE 1.

Titration of cCTnC·2Ca²⁺ with EGCg. Two-dimensional ¹H,¹⁵N HSQC (a) and ¹H,¹³C-HSQC (b) spectra arising from backbone and side chain amide groups (a) and side chain methyl groups (b) are overlaid for a series of EGCg additions. Each titration point represents the titration points described under “Experimental Procedures.” The titration was made into ¹³C,¹⁵N-labeled cCTnC·2Ca²⁺, and both the ¹H,¹⁵N HSQC and ¹H,¹³C-HSQC spectra were acquired at each titration point. Assignments of some of the cross-peaks are labeled. The multiple contours (●) represent the initial point in the titration, with no EGCg added, and each open contour (○) represents a specific point in the titration for a given residue. b, red contours represent cross-peaks with negative intensity, a feature of the constant time ¹H,¹³C-HSQC experiment. The direction the peaks shift is indicated with arrows, for example see Gly¹²⁵. c, curves represent the amide resonances belonging to residues affected by ligand binding, as shown in a. The curves were fit as a function of normalized total chemical shift perturbation versus [EGCg]_total/[cCTnC·2Ca²⁺]_total. The total chemical shift changes were calculated in hertz as follows: Δδ = ((Δδ¹H)² + (Δδ¹⁵N)²)^1/2. Because hertz is used instead of parts/million, a correction factor of 1/5 for the ¹⁵N dimension is not used. d, chemical shift mapping on the structure of cCTnC·2Ca²⁺. The ribbon representation of cCTnC·2Ca²⁺ is shown in yellow, and residues that were perturbed greater than the mean chemical shift change for all backbone amide resonances of cCTnC are colored in red.

FIGURE 2.

Assignment of EGCg. a, chemical structure of EGCg. The benzenediol is labeled as ring A, the pyrogallol ring as B, the galloyl moiety as B′, and ring C is the tetrahydropyran moiety. The hydrogen atoms attached to carbon atoms are also labeled. b, assigned one-dimensional ¹H NMR spectrum of EGCg in DMSO-d₆. c, a few strip plots from the two-dimensional NOESY with resonances assigned that belong to EGCg in complex with cCTnC·2Ca²⁺. The data were acquired in D₂O as to remove amide signals that predominate this region of the two-dimensional NOESY spectrum in H₂O. Details of the experiment are outlined in Table 1.

FIGURE 3.

Intermolecular NOEs between EGCg and cCTnC·2Ca²⁺. a, series of strip plots assigned from the two-dimensional ¹³C-edited/filtered NOESY NMR experiment. The ¹H resonances that correspond to EGCg are labeled on the right side of the spectra, and the ¹H that correspond to ¹⁵N,¹³C-labeled cCTnC·2Ca²⁺ are labeled at the top of the strips plots. The circled peak is an artifact from the intermolecular NOE experiment. b, schematic depiction of several of the NOE contacts assigned in a. EGCg is shown in stick representation with carbon atoms colored in purple, oxygen atoms color in red, and hydrogen atoms colored in white. cCTnC is depicted in schematic representation with the residues involved in making NOEs to EGCg shown in stick representation. Carbon atoms for cCTnC are colored in gray, sulfur atoms in yellow, and hydrogen atoms in white. The dotted lines indicate contacts measured by the ¹³C-edited/filtered NOESY NMR experiment.

FIGURE 4.

Diagram of the solution structure of cCTnC·2Ca²⁺·EGCg. a, ensemble of the 30 lowest energy structures of EGCg in association with cCTnC·2Ca²⁺ is depicted with just the backbone atoms of cCTnC drawn as ribbons and of EGCg as sticks. The ensemble of cCTnC·2Ca²⁺ is colored in gray; the Ca²⁺ ions are shown as gray spheres, and the ensemble of EGCg is colored with carbon atoms in purple and oxygen atoms in red. b, 90° rotation about the y axis. c, ensemble of EGCg and the chemical groups of EGCg are labeled. d, schematic representation of the lowest energy structure of the ensemble of cCTnC·2Ca²⁺·EGCg with the helices labeled and consistent coloring as above. The orientation of cCTnC is the same as in a.

FIGURE 5.

A backbone overlay of cCTnC·2Ca²⁺·EGCg with several structures of cCTnC. In all of the images, cCTnC is depicted in schematic form and colored in gray. EGCg is shown in stick representation, and carbon atoms are colored in purple; oxygen atoms are colored in red; and hydrogen atoms are shown in white. a, cCTnC in the cCTnC·2Ca²⁺·EGCg complex is overlaid with cCTnC in the cCTnC·2Ca²⁺ complex (PDB 3CTN) shown in magenta. b, 90° rotation about the y axis. The helices are labeled in two diagrams, and EGCg is also labeled. c, cCTnC in the cCTnC·2Ca²⁺·EGCg complex is overlaid with cCTnC in the cCTnC·2Ca²⁺·EMD 57033 complex (PDB 1IH0) shown in orange. EMD 57033 is colored with carbon atoms shown in green, sulfur atoms in yellow, oxygen atoms in red, and hydrogen atoms in white. d, 90° rotation about the y axis. EMD 57033 is identified with an arrow. e, cCTnC in the cCTnC·2Ca²⁺·EGCg complex is overlaid with cCTnC in the cCTnC·2Ca²⁺·cTnI-(34–71) complex (PDB 1J1D) shown in lime green. f, 90° rotation about the y axis. cTnI-(34–71) is labeled in both representations.

FIGURE 6.

Titration of cCTnC·2Ca²⁺·cTnI-(34–71) with EGCg. Two-dimensional ¹H,¹⁵N HSQC (a) and ¹H,¹³C-HSQC (b) spectra arising from backbone and side chain amide groups (a) and side chain methyl groups (b) are overlaid for a series of EGCg additions. Each titration point represents the titration points described under “Experimental Procedures.” The titration was made into ¹³C,¹⁵N-labeled cCTnC·2Ca²⁺·cTnI-(34–71), and both the ¹H,¹⁵N HSQC and ¹H,¹³C-HSQC spectra were acquired at each titration point. Assignments of some of the cross-peaks are labeled. The multiple contours (●) represent the initial point in the titration, with no EGCg added, and the open contours (○) represent the end point in the titration for a given residue. b, red contours represent cross-peaks with negative intensity, a feature of the constant time ¹H,¹³C-HSQC experiment. The direction that the peaks shift is indicated by arrows, for example see Met¹²⁰. c, curves represent a number of residues affected by ligand binding, as shown in a. The curves were fit as a function of normalized total chemical shift perturbation versus [EGCg]_total/[cCTnC·2Ca²⁺·cTnI-(34–71)]_total. d, cCTnC·2Ca²⁺·cTnI-(34–71) complex is shown in lime green with cCTnC·2Ca²⁺ and cTnI-(34–71) shown in schematic representation. Chemical shift perturbations of the backbone amide resonances induced by EGCg binding to cCTnC·2Ca²⁺·cTnI-(34–71) are colored in red for residues that shifted greater than the mean shift of all residues of cCTnC. Total chemical shift changes are calculated in hertz as follows: Δδ = ((Δδ¹H)² + (Δδ¹⁵N)²)^1/2. Because hertz is used instead of parts/million, a correction factor of 1/5 for the ¹⁵N dimension is not used.