Investigating the effects of tropomyosin mutations on its flexibility and interactions with filamentous actin using molecular dynamics simulation

Abstract

Tropomyosin (Tpm) is a two-chained α-helical coiled-coil protein that binds to filamentous actin (F-actin), and regulates its interactions with myosin by occupying three average positions on F-actin (blocked, closed, and open). Mutations in the Tpm are linked to heart diseases including hypertrophic cardiomyopathy (HCM) and dilated cardiomyopathy (DCM). To elucidate the molecular mechanisms of Tpm mutations (including DCM mutation E54K, HCM mutations E62Q, A63V, K70T, V95A, D175N, E180G, L185R, E192K, and a designed synthetic mutation D137L) in terms of their effects on Tpm flexibility and its interactions with F-actin, we conducted extensive molecular dynamics simulations for the wild-type and mutant Tpm in complex with F-actin (total simulation time 160 ns per mutant). The mutants exhibited distinct changes (i.e., increase or decrease) in the overall and local flexibility of the Tpm coiled-coil, with each mutation causing both local and long-range modifications of the Tpm flexibility. In addition, our binding calculations revealed weakened Tpm-F-actin interactions (except for L185R, D137L and A63V) involving five periods of Tpm, which correlate with elevated fluctuation of Tpm relative to the blocked position on F-actin that may lead to easier activation and increased Ca-sensitivity. We also simulated the αβ/βα-Tpm heterodimer in comparison with the αα-Tpm homodimer, which revealed greater flexibility and weaker actin binding in the heterodimer. Our findings are consistent with a complex mechanism underlying how different Tpm mutations perturb the Tpm function in distinct ways (e.g., by affecting specific sites of Tpm), which bear no simple links to the disease phenotypes (e.g., HCM vs. DCM).

The Relaxation Properties of Myofibrils Are Compromised by Amino Acids that Stabilize α-Tropomyosin

“…Among Tm noncanonical amino acids, recent studies have examined two highly conserved residues in the middle part of the aaTm molecule: D137 (a charged residue in a d hydrophobic position) and G126 (a small, neutral residue in a g charged position). Replacement of these residues with canonical ones, D137L and G126R (9,10), was shown in solution studies to significantly increase coiled-coil stability and decrease molecular mobility or flexibility (9,(11)(12)(13). A significant decrease in the flexibility of reconstructed thin filaments with the combined D137L/G126R substitution was also shown by means of the two-bead optical trap technique (14).…”

Insights and Challenges of Multi-Scale Modeling of Sarcomere Mechanics in cTn and Tm DCM Mutants—Genotype to Cellular Phenotype

“…A 2012 study used MD to study familial HCM associated Tm mutations D175N and E180G and found that both mutations lead to increased flexibility of Tm and therefore, decreased persistence length of the molecule (Li et al, 2012). Another MD study has also been performed on HCM Tm mutations E62Q, A63V, K70T, V95A, D175N, E180G, L185R, E192K in order to explore the effects of point mutations on Tm flexibility and Tm-actin interactions (Zheng et al, 2016). In the case of DCM, a time-independent electrostatic snapshot of the DCM associated Tm mutation showed that E54K and E40K mutations alter the surface charge of Tm, which may affect Tm-actin interaction (Olson et al, 2001; Chang et al, 2014).…”

“…In the case of DCM, a time-independent electrostatic snapshot of the DCM associated Tm mutation showed that E54K and E40K mutations alter the surface charge of Tm, which may affect Tm-actin interaction (Olson et al, 2001; Chang et al, 2014). A time-dependent MD study was also performed on the E54K Tm mutant in combination with 7 actin monomers and showed that this mutation causes increased stiffness and decreased curvature in the Tm molecule overall while stabilizing and destabilizing the coiled coil structure in different regions of the molecule and greatly weakening Tm-actin binding (Zheng et al, 2016). More MD studies are needed in the area of Tm DCM mutations in order to visualize effects of other mutations, such as E40K, that may affect Tm-actin and Tm-cTn interactions.…”

Predicting Effects of Tropomyosin Mutations on Cardiac Muscle Contraction through Myofilament Modeling

“…However, recent work has shown that point mutations in tropomyosin can also affect the energy landscape of tropomyosin interactions with actin, providing another potential route for mutations to affect thin filament calcium regulation (Orzechowski et al, 2014a,b). Altered actin binding by mutant tropomyosin is also supported by numerous experimental and computational studies (Boussouf et al, 2007; Janco et al, 2012; Zheng et al, 2016). The surface interactions of tropomyosin and actin may ultimately affect myosin crossbridge formation, as suggested by length perturbation analysis studies (Bai et al, 2011).…”

“…This suggests that mutation effects are not confined to stiffness alone and that other molecular mechanisms should be considered. Indeed, tropomyosin mutants have previously been determined to alter the interactions between tropomyosin and the actin surface (Orzechowski et al, 2014a; Zheng et al, 2016), which may in turn affect both the blocked-to-closed and closed-to-open (myosin-induced) transitions of tropomyosin across actin. We therefore entertained the possibility that introducing changes to the BC equilibrium constant and duty cycle in addition to the assumed stiffness changes could produce a reasonable fit to mutant data.…”