Phosphorylation-induced distance change in a cardiac muscle troponin I mutant.
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.
Dong
W J
WJ
Department of Biochemistry and Molecular Genetics, University of Alabama at Birmingham, 35294-2041, USA.
Chandra
M
M
Xing
J
J
She
M
M
Solaro
R J
RJ
Cheung
H C
HC
D000818:Animals; D002118:Calcium / Q000494:pharmacology; D002417:Cattle; D055598:Chemical Phenomena; D002627:Chemistry, Physical; D004533:Egtazic Acid / Q000494:pharmacology; D005456:Fluorescent Dyes; D008274:Magnesium / Q000494:pharmacology; D008433:Mathematics; D009154:Mutation; D009206:Myocardium / Q000737:chemistry*; D009282:Naphthalenesulfonates; D010766:Phosphorylation; D010768:Phosphoserine / Q000378:metabolism*; D013050:Spectrometry, Fluorescence; D013329:Structure-Activity Relationship; D013816:Thermodynamics; D019209:Troponin C / Q000378:metabolism; D019210:Troponin I / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism
D005456:Fluorescent Dyes; D009282:Naphthalenesulfonates; D019209:Troponin C; D019210:Troponin I; D010768:Phosphoserine; D004533:Egtazic Acid; C011488:IAANS; D008274:Magnesium; D002118:Calcium