Phosphorylation-Induced Distance Change in a Cardiac Muscle Troponin I Mutant

Abstract

Phosphorylation of two adjacent serine residues in the unique N-terminal extension of cardiac muscle troponin I (cTnI) is known to decrease the Ca2+-sensitivity of cardiac myofilaments. To probe the structural significance of the N-terminal extension, we have constructed two cTnI mutants each containing a single cysteine: (1) a full-length cTnI mutant (S5C/C81I/C98S) and (2) a truncated cTnI mutant (S9C/C50I/C67S) in which the N-terminal 32 amino acid residues were deleted. We determined the apparent binding constants for the complex formation between IAANS-labeled cardiac troponin C (cTnC) and the two cTnI mutants. The affinities of the cTnC for the truncated cTnI mutant were: (1) 1.5 x 10(6) M(-1) in EGTA, (2) 28.9 x 10(6) M(-1) in Mg2+, and (3) 87.5 x 10(6) M(-1) in Mg2+ + Ca2+. These binding constants were approximately 1.4-fold smaller than the corresponding values obtained with the full-length cTnI mutant, suggesting a very small contribution of the N-terminal extension to the binding of cTnI to cTnC. Cys-5 in the full-length cTnI mutant was labeled with IAANS, and the distribution of the separation between this site and Trp-192 was determined by analysis of the efficiency of fluorescence resonance energy transfer from Trp-192 to IAANS. The following mean distances were obtained with the unphosphorylated full-length mutant: 44.4 A (cTnI alone), 48.3 A (cTnI + cTnC), 46.3 A (cTnI + cTnC in Mg2+), and 51.6 A (cTnI + cTnC in Mg2+ + Ca2+). The corresponding values of the mean distance determined with the phosphorylated full-length cTnI mutant were 35.8, 36.6, 34.8, and 37.3 A. The phosphorylation of cTnI reduced the half-width of the distribution from 9.5 to 3.7 A. Similar but less pronounced decreases of the half-widths were also observed with the phosphorylated cTnI complexed with cTnC in different ionic conditions. Thus, phosphorylation of cTnI resulted in a decrease of 9-12 A in the mean distance between the sites located at the N- and C-terminal portion of cTnI. Our results indicate that phosphorylation elicits a change in the conformation of cTnI which underlies the basis of the phosphorylation-induced modulation of cTnI activity.

The unique functions of cardiac troponin I in the control of cardiac muscle contraction and relaxation

“…In the model, the acidic amino terminus of the N-extension interacts with basic residues within the inhibitory region of cTnI. The phosphorylation-induced bending of cTnI is consistent with biochemical studies showing that the axial ratio decreases upon phosphorylation and an asymmetrical to a more symmetrical change in shape consistent with a shorter, broader structure [54][55][56].…”

Invited review: Probing the structures of muscle regulatory proteins using small‐angle solution scattering

“…Four years later, after the publication of the crystal structure of the cTn core, this characteristic bending of TnI was confirmed by solution NMR studies. 52 A variety of studies using fluorescence, photochemical cross-linking, FRET, NMR, and crystallography provided key contact information within the cTn complex 40,[53][54][55][56][57][58][59] such that the SANS model of the ternary cTn could then be further interpreted with regards to subunit arrangements. For example, the lower lobe of the cTnC in the SANS model represents the C-terminal domain (see Figure 3).…”

Effects of PKA Phosphorylation of Cardiac Troponin I and Strong Crossbridge on Conformational Transitions of the N-Domain of Cardiac Troponin C in Regulated Thin Filaments

“…The FRET donor decays determined in the presence of acceptor were analyzed using global analysis software (GlobeCurve) as previously described (35). This procedure yielded a distribution of inter-site distances (21). The distance at the peak of the distribution was taken as the mean distance between donor and acceptor sites.…”