Effects of T142 phosphorylation and mutation R145G on the interaction of the inhibitory region of human cardiac troponin I with the C-domain of human cardiac troponin C.
Cardiac troponin I (cTnI) is the inhibitory component of the troponin complex, and its interaction with cardiac troponin C (cTnC) plays a critical role in transmitting the Ca(2+) signal to the other myofilament proteins in heart muscle contraction. The switch between contraction and relaxation involves a movement of the inhibitory region of cTnI (cIp) from cTnC to actin-tropomyosin. This region of cTnI is prone to missense mutations in heart disease, and a specific mutation, R145G, has been associated with familial hypertrophic cardiomyopathy. It also contains the unique cardiac PKC phosphorylation site at residue T142. To determine the structural consequences of the mutation R145G and the T142 phosphorylation on the interaction of cIp with cTnC, we have utilized 2D [(1)H, (15)N]-HSQC NMR spectroscopy to monitor the binding of native cIp, cIp-R (R145G), and cIp-P (phosphorylated T142), respectively, to the Ca(2+)-saturated C-domain of cTnC (cCTnC.2Ca(2+)). We also report a strategy for cloning, expression, and purification of cTnI peptide, and both synthetic and recombinant peptides are used in this study. NMR chemical shift mapping indicates that the binding epitope of cIp on cCTnC.2Ca(2+) is not greatly affected, but the affinity is reduced by approximately 14-fold by the T142 phosphorylation and approximately 4-fold by the mutation R145G, respectively. This suggests that these modifications of cIp have an adverse effect on the binding of cIp to cCTnC.2Ca(2+). These perturbations may correlate with the impairment or loss of cTnI function in heart muscle contraction.
Lindhout
Darrin A
DA
CIHR Group in Protein Structure and Function, Department of Biochemistry, University of Alberta, Edmonton, Alberta, Canada T6G 2H7.
Li
Monica X
MX
Schieve
Dean
D
Sykes
Brian D
BD
D000595:Amino Acid Sequence; D001120:Arginine / Q000235:genetics*; D002118:Calcium / Q000378:metabolism; D002413:Cations, Divalent / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics; D005998:Glycine / Q000235:genetics*; D006801:Humans; D008969:Molecular Sequence Data; D020125:Mutation, Missense; D009200:Myocardial Contraction / Q000235:genetics; D009206:Myocardium / Q000378:metabolism*; D019906:Nuclear Magnetic Resonance, Biomolecular; D010446:Peptide Fragments / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D010766:Phosphorylation; D011485:Protein Binding / Q000235:genetics; D011493:Protein Kinase C / Q000378:metabolism; D017434:Protein Structure, Tertiary / Q000235:genetics; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000302:isolation & purification / Q000378:metabolism; D013912:Threonine / Q000378:metabolism*; D019209:Troponin C / Q000378:metabolism*; D019210:Troponin I / Q000235:genetics / Q000378:metabolism* / Q000494:pharmacology*
D002413:Cations, Divalent; D010446:Peptide Fragments; D011994:Recombinant Proteins; D019209:Troponin C; D019210:Troponin I; D013912:Threonine; D001120:Arginine; D011493:Protein Kinase C; D002118:Calcium; D005998:Glycine