Using lanthanide ions to align troponin complexes in solution: order of lanthanide occupancy in cardiac troponin C

Abstract

The potential for using paramagnetic lanthanide ions to partially align troponin C in solution as a tool for the structure determination of bound troponin I peptides has been investigated. A prerequisite for these studies is an understanding of the order of lanthanide ion occupancy in the metal binding sites of the protein. Two-dimensional [(1)H, (15)N] HSQC NMR spectroscopy has been used to examine the binding order of Ce(3+), Tb(3+), and Yb(3+) to both apo- and holo-forms of human cardiac troponin C (cTnC) and of Ce(3+) to holo-chicken skeletal troponin C (sTnC). The disappearance of cross-peak resonances in the HSQC spectrum was used to determine the order of occupation of the binding sites in both cTnC and sTnC by each lanthanide. For the lanthanides tested, the binding order follows that of the net charge of the binding site residues from most to least negative; the N-domain calcium binding sites are the first to be filled followed by the C-domain sites. Given this binding order for lanthanide ions, it was demonstrated that it is possible to create a cTnC species with one lanthanide in the N-domain site and two Ca(2+) ions in the C-domain binding sites. By using the species cTnC.Yb(3+).2 Ca(2+) it was possible to confer partial alignment on a bound human cardiac troponin I (cTnI) peptide. Residual dipolar couplings (RDCs) were measured for the resonances in the bound (15)N-labeled cTnI(129-148) by using two-dimensional [(1)H, (15)N] inphase antiphase (IPAP) NMR spectroscopy.

Figure 1.

Portions of a 2D {¹H, ¹⁵N} HSQC spectra of ¹⁵N-cTnC saturated with Ca²⁺ (A), subsequently titrated to one molar equivalent with Ce³⁺ (B), Tb³⁺ (C), or Yb³⁺ (D). Asterisks mark the position of N-domain binding site II resonances that have either shifted or broadened beyond detection after the addition of one molar equivalent of lanthanide to holo-cTnC.

Figure 2.

Portions of a 2D {¹H, ¹⁵N} HSQC spectra of ¹⁵N-cTnC•Ce³⁺•2Ca²⁺ depicting the downfield Gly residues of the C-domain binding loops III and IV. This series demonstrates the binding order of Ce³⁺ to the C-domain sites after the binding of one molar equivalent of Ce³⁺ to holo-cTnC. The titration progresses from the upper left box at 1.1 eq and proceeds to the lower right by 0.1 eq steps. The two C-domain Gly residues are labeled, and an additional unassigned residue is labeled with a question mark. This residue moved into this region upon the binding of the first equivalent of Ce³⁺ and is likely an N-domain resonance.

Figure 3.

2D {¹H, ¹⁵N} HSQC spectrum of both ¹⁵N-apo-cTnC (A) and ¹⁵N-cTnC bound to one molar equivalent of Ce³⁺ (B). Arrows point to the apo-positions of the site II binding loop resonances in both spectra.

Figure 4.

Portions of a 2D {¹H, ¹⁵N} HSQC spectrum showing several points in the titration of 0.92 mM ¹⁵N-sTnC with Ce³⁺. The area containing downfield Gly residues of the ion binding loops is expanded, and the panels follow through the titration from apo-saturated (A) to Ca²⁺-saturated (B) through one, two, three, and four molar equivalents (C–F, respectively). All visible residues are labeled. Note that the splitting in the N-domain Gly resonances is likely due to Cys cross-bridge formation.

Figure 5.

Expanded region of 1D NMR spectra at 600 MHz (A) and 800 MHz (B) of unlabeled cTnC, as well as a 2D {¹H, ¹⁵N} IPAP spectrum also at 800 MHz (C) of ¹⁵N-cTnI_129–148 bound to unlabeled cTnC. (A, B) The successful addition of Yb³⁺ solely to the N-domain site II, with the lower spectrum in each depicting the downfield Gly residues of cTnC•3Ca²⁺, and the upper spectrum depicting the same region of cTnC•Yb³⁺•2Ca²⁺. (C) The splitting of the ¹⁵N-cTnI_129–148 resonances and the measured RDCs for each residue. For each pair of resonances connected by a dashed line, the upper resonance is the inphase resonance and the lower resonance is antiphase.

Figure 5.

Expanded region of 1D NMR spectra at 600 MHz (A) and 800 MHz (B) of unlabeled cTnC, as well as a 2D {¹H, ¹⁵N} IPAP spectrum also at 800 MHz (C) of ¹⁵N-cTnI_129–148 bound to unlabeled cTnC. (A, B) The successful addition of Yb³⁺ solely to the N-domain site II, with the lower spectrum in each depicting the downfield Gly residues of cTnC•3Ca²⁺, and the upper spectrum depicting the same region of cTnC•Yb³⁺•2Ca²⁺. (C) The splitting of the ¹⁵N-cTnI_129–148 resonances and the measured RDCs for each residue. For each pair of resonances connected by a dashed line, the upper resonance is the inphase resonance and the lower resonance is antiphase.

Figure 6.

A plot of the absolute value of measured RDCs from ¹⁵N-cTnI_129–148 at 800 MHz versus the amino acid sequence of ¹⁵N-cTnI_129–148. The inset structure of bound ¹⁵N-cTnI_129–148 to the C-domain of cTnC is taken from Lindhout et al. 2002.