Structural Transition of N-Terminal Extension of Cardiac Troponin-I Complexed with Troponin-C caused by PKC-Mediated Phosphorylation, as Revealed by Spin Labeling EPR

Abstract

Cardiac troponin (cTn) complex consisting of cTnI, cTnC and cTnT is responsible for cardiac mechanotransduction. Aberrent phosophorylation of cTnI by PKC on S42/44 or its phosphorylation mimic mutant S42/44D decreases Ca2+ sensitivity and tension force, consequently generating heart failure. Until now, consequence of cTnI by PKC phosphorylation on molecular level and associated structural mechanisms involved in it are still elusive. Combined with previously published NMR spectra and our bioinformatics analysis, this distinct N-extension of cTnI is strongly shown to be intrinsically disordered. To simulate the possible conformation of this intrinsically disordered region and potential conformational transition caused by PKC when binding with cTnC, Monte Carlo simulation was implemented to explore potential docking sites on cTnC, of which all the predicted structures were evaluated by total and binding energy with the criteria under RosettaCommons software suite. We screened around 10,000 structures for cTnC-I and cTnC-I (S42/44D) complexes, respectively. For cTnC-I complex, flexible N-extension of cTnI docked on N-domain of cTnC, putatively assisting to stabilize the full-open state of cTnC which binds strongly the regulatory region of cTnI to initiate muscle contraction. In contrast, as for cTnC-I (S42/44D), it localized on C-domain of cTnC, thus deficiently to maintain full-open state of cTnC, causing a weak interaction between the regulatory region of cTnI (S42/44D) and cTnC to generate heart failure. To verify our prediction, we are measuring residual mobility and inter-residual distance from spin labels placed on cTnI or cTnI (S42/44D) and cTnC. Preliminary mobility measurement of MSL-labeled cTnC(C84) indicated loose interaction between the regulatory region of cTnI (S42/44D) and cTnC. Measurements on the other sites are in progress.