NMR and mutagenesis studies on the phosphorylation region of human cardiac troponin I.
Phosphorylation of the cardiac troponin complex by PKA at S22 and S23 of troponin I (TnI) accelerates Ca(2+) release from troponin C (TnC). The region of TnI around the bisphosphorylation site binds to, and stabilizes, the Ca(2+) bound N-terminal domain of TnC. Phosphorylation interferes with this interaction between TnI and TnC resulting in weaker Ca(2+) binding. In this study, we used (1)H-(15)N-HSQC NMR to investigate at the atomic level the interaction between an N-terminal fragment of TnI consisting of residues 1-64 of TnI (I1-64) and TnC. We produced several mutants of I1-64, TnI, and TnC to test the contribution of certain residues to the transmission of the phosphorylation signal in both NMR experiments and functional assays. We also investigated how phosphorylation of the PKC sites in I1-64 (S41 and S43) affects the interaction of I1-64 with TnC. We found that phosphorylation of S22 and S23 produced only localized effects in the structure of I1-64 between residues 24 and 34. Residues 1-17 of I1-64 did not bind to TnC, and residues 38-64 bound tightly to the C-terminal domain of TnC regardless of phosphorylation. Residues 22-34 bound weakly to TnC in a phosphorylation sensitive manner. Bisphosphorylation prevented this phosphorylation switch region from interacting with TnC. Systematic mutation of residues in the phosphorylation switch did not prevent PKA phosphorylation from accelerating Ca(2+) release from troponin. We conclude that the phosphorylation switch binds to TnC via an extended interaction site spanning residues R19 to A34.
Ward
Douglas G
DG
School of Biosciences, University of Birmingham, Edgbaston, Birmingham B15 2TT, United Kingdom.
Brewer
Susan M
SM
Gallon
Clare E
CE
Gao
Yuan
Y
Levine
Barry A
BA
Trayer
Ian P
IP
D000409:Alanine / Q000235:genetics; D001120:Arginine / Q000235:genetics; D002118:Calcium / Q000378:metabolism; D006801:Humans; D046911:Macromolecular Substances; D016297:Mutagenesis, Site-Directed; D009206:Myocardium / Q000201:enzymology / Q000378:metabolism*; D009587:Nitrogen Isotopes / Q000378:metabolism; D019906:Nuclear Magnetic Resonance, Biomolecular; D010446:Peptide Fragments / Q000235:genetics / Q000378:metabolism; D010766:Phosphorylation; D011485:Protein Binding / Q000235:genetics; D011493:Protein Kinase C / Q000378:metabolism; D051571:Protein Kinase C-alpha; D017434:Protein Structure, Tertiary / Q000235:genetics; D011522:Protons; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D012694:Serine / Q000235:genetics; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D019209:Troponin C / Q000235:genetics / Q000378:metabolism; D019210:Troponin I / Q000235:genetics* / Q000378:metabolism*
D046911:Macromolecular Substances; D009587:Nitrogen Isotopes; D010446:Peptide Fragments; D011522:Protons; D011994:Recombinant Proteins; D019209:Troponin C; D019210:Troponin I; D012694:Serine; D001120:Arginine; C496628:PRKCA protein, human; D011493:Protein Kinase C; D051571:Protein Kinase C-alpha; D000409:Alanine; D002118:Calcium