The Importance of Intrinsically Disordered Segments of Cardiac Troponin in Modulating Function by Phosphorylation and Disease-Causing Mutations

Abstract

Troponin plays a central role in regulation of muscle contraction. It is the Ca2+ switch of striated muscles including the heart and in the cardiac muscle it is physiologically modulated by PKA-dependent phosphorylation at Ser22 and 23. Many cardiomyopathy-related mutations affect Ca2+ regulation and/or disrupt the relationship between Ca2+ binding and phosphorylation. Unlike the mechanism of heart activation, the modulation of Ca2+-sensitivity by phosphorylation of the cardiac specific N-terminal segment of TnI (1–30) is structurally subtle and has proven hard to investigate. The crystal structure of cardiac troponin describes only the relatively stable core of the molecule and the crucial mobile parts of the molecule are missing including TnI C-terminal region, TnI (1–30), TnI (134–149) (“inhibitory” peptide) and the C-terminal 28 amino acids of TnT that are intrinsically disordered. Recent studies have been performed to answer this matter by building structural models of cardiac troponin in phosphorylated and dephosphorylated states based on peptide NMR studies. Now these have been updated by more recent concepts derived from molecular dynamic simulations treating troponin as a dynamic structure. The emerging model confirms the stable core structure of troponin and the mobile structure of the intrinsically disordered segments. We will discuss how we can describe these segments in terms of dynamic transitions between a small number of states, with the probability distributions being altered by phosphorylation and by HCM or DCM-related mutations that can explain how Ca2+-sensitivity is modulated by phosphorylation and the effects of mutations.

Troponin structure and function: a view of recent progress

“…Two molecular dynamics studies of the troponin core have addressed this mutation. The study of Lindert et al (2015) may have been compromised by the size of the box used for simulation (Papadaki and Marston 2016;Zamora et al 2016). However, a study by Dvornikov et al (2016) used a larger box, although simulation times were still short (100 ns).…”

“…Recently the structure of the cardiac troponin core has been investigated by molecular dynamics simulations (Cheng et al 2014;Papadaki and Marston 2016;Zamora et al 2016); (Fig. 3).…”

Molecular defects in cardiac myofilament Ca2+- regulation due to cardiomyopathy-linked mutations can be reversed by small molecules binding to troponin

“…Although structural determinations of ligand binding (EGCG, W-7) to troponin subunits have been made, most studies have involved binary complexes (TnI and TnC) and partial sequences (Robertson et al, 2010;Botten et al, 2013;Hwang and Sykes, 2015). Recent molecular dynamics simulations have emphasized the importance of studying the intact troponin core with TnI, TnC, and TnT present (Papadaki and Marston, 2016;Zamora et al, 2016). Moreover, studies on the docking of EGCG, Silybin A and Silybin B by molecular dynamics simulations suggest that these compounds bind and crosslink the N-terminal peptide of TnI in the vicinity of the phosphorylatable serines22 and−23 to the N terminal domain of TnC and that the three compounds have different dynamics.…”

“…It is notable that the phosphorylatable N-terminus and the "switch peptide" of TnI, critical for Ca 2+ -regulation, are intrinsically disordered peptides (Hwang et al, 2014;Papadaki and Marston, 2016). By analogy with similar disordered peptides containing regulatory phosphorylation sites it is suggested that there may be negligible differences in time-averaged structures between phosphorylated and unphosphorylated states, but rather phosphorylation induces a change in protein dynamics from an ordered to a less ordered state (Colson et al, 2012).…”

“…Whilst the structural mechanism of the troponin Ca 2+ switch is well-documented (Takeda et al, 2003), the structural basis of lusitropy is poorly understood since both the phosphorylated N-terminal peptide and the regulatory "switch peptide" of TnI are intrinsically disordered, however recent studies using molecular dynamics simulations have begun to explain this phenomenon (Papadaki and Marston, 2016). The study of this phosphorylation dependent regulation has been recently stimulated by the discovery that a primary effect of many mutations in thin filament proteins associated with cardiomyopathy is the uncoupling of this relationship (Bayliss et al, 2012;Memo et al, 2013;Messer and Marston, 2014).…”