NMR and Mutagenesis Studies on the Phosphorylation Region of Human Cardiac Troponin I

Abstract

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.

Impact of site-specific phosphorylation of protein kinase A sites Ser23and Ser24of cardiac troponin I in human cardiomyocytes

“…ine, respectively. Previous studies have indicated that this is indeed the case with respect to interactions between the cTn subunits (12,29), Ca 2ϩ dissociation rates from cTnC (45), and pCa 50 (10). Moreover, it can be noted that we observed similar functional parameters in cells exchanged with dephosphorylated cTnI mimicked with alanine substitutions at Ser 23 and Ser 24 and unphosphorylated wild-type cTnI (Table 2).…”

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 structural consequence of phosphorylation of cTnI on protein-protein interactions that occur within the cTn complex as well as conformational changes of cTnI have been extensively studied using NMR (22,23,26,39,40) and FRET (21,33,(41)(42)(43). These studies suggest that the N-terminal extension of cTnI, most likely the residues immediately before the PKA phosphorylation sites, interact with the N-domain of cTnC.…”

Protein Phosphorylation and Signal Transduction in Cardiac Thin Filaments

“…Ward et al (53) also showed that peptide residues 1-18 of cTnI do not bind to cTnC. NMR studies (53,54), as well as previous studies (37), demonstrated that PKA phosphorylation at Ser 23 and Ser 24 produced only localized structural effects in segment residues ϳ25-35.…”