Molecular Dynamics Studies on Phosphorylated and Unphosphorylated Cardiac Troponin

Abstract

The Ca2+-sensitivity of cardiac muscle is modulated by a phosphorylation-dependent interaction between the N-terminal peptide of troponin I (TnI 1-30) and the N-terminal lobe of troponin C (TnC). This interaction enhances Ca2+-sensitivity and is abolished by PKA phosphorylation of Ser 22 and 23.The sequences of TnI involved are missing in X-ray diffraction structures. NMR has been used to define the structure of the missing peptides based on their binary complexes. Molecular dynamics (MD) offers a new approach that can analyze the entire troponin structure.We have applied MD simulations on the Takeda et al. structure of the core domain of human cardiac troponin in the Ca2+ saturated form. Simulations have been performed in explicit water and on an expanded model of the full crystallographic structure (385 aa). All simulations have been performed for at least 1µs with the AMBER GPU MD package in an isobaric-isothermal, NPT, ensemble. The first 31 residues of TnI, were modeled in as a linear chain according to the Howarth model. Simulations were run firstly of unphosphorylated troponin for 1µs followed by phosphorylation of the 1µs structure. After 1µs, further dephosphorylation and rephosphorylation simulations were run to check reversibility.After 1µs of simulation the crystal structure of unphosphorylated troponin is retained and persistent conformations of the modelled-in peptides stabilize with 1-30 looping over TnC N-terminal domain making weak contacts in the Ser22/23 region. Upon phosphorylation only the N-terminal domain of TnC changed, notably slight helix reorientation and a reduced interaction of cTnC 1-80 with TnI (1-30) and the switch peptide (144-160). The orientation of TnC N-terminus relative to the IT arm changed and became more mobile. RMSF plots pinpointed the amino acids involved in these interactions and the effect on Ca2+-binding.