Abstract
During β-adrenergic stimulation, cTnI is phosphorylated by PKA at sites S23/S24, located at the N-terminus of cTnI. This phosphorylation has been shown to decrease KCa, decrease pCa50, and weaken C-I interaction. However, as the N-terminus of cTnI (residues 1-40) has not been resolved, the structural basis of how PKA phosphorylation influences cTn structure and calcium binding still remains elusive. Here, for the first time, we built up the cTn complex structure (including residues cTnC 1-161, cTnI 1-172, and cTnT 236-285) based on Rosevear's NMR structure. To mimic phosphorylation status, we constructed a bis-phosphomimics model by mutating S23/S24 of cTnI to aspartic acid. Then, 150 ns duplicated molecular dynamics (MD) simulations were performed on both WT and TnI-S23DS24D models. Through residue-residue contact analysis, we found that introducing these two phosphomimic mutations to sites S23/S24 significantly alters the native C-I interaction, except in the switch/inhibitory regions of cTnI. This phosphorylation also leads to the formation of intra-subunit interaction between N-terminus and inhibitory peptide of cTnI. Then, we studied how this phosphorylation influences the calcium handling in site II by calculating the distance between calcium and its coordinating residues. We saw that Ca2+ could coordinate with four residues (D65, D67, D73, E76), and S69 is the most flexible residue, which is in agreement with our previous observation. We also analyzed the interhelical angle and distance between A/B and B/C helices and quantified the exposure of hydrophobic surface in cTnC related to cTnI switch peptide binding. Simulations of an entire cTn complex model and its bis-phosphomimics status can elucidate the dynamic interplay among cTnC, cTnI and cTnT subunits, as well as the cellular progress during β-adrenergic stimulation on a molecular level. HL65497, HL11197 (MR), 8P41GM103426 (AM & AM).
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have