Electrostatic interaction map reveals a new binding position for tropomyosin on F-actin

Abstract

Azimuthal movement of tropomyosin around the F-actin thin filament is responsible for muscle activation and relaxation. Recently a model of αα-tropomyosin, derived from molecular-mechanics and electron microscopy of different contractile states, showed that tropomyosin is rather stiff and pre-bent to present one specific face to F-actin during azimuthal transitions. However, a new model based on cryo-EM of troponin- and myosin-free filaments proposes that the interacting-face of tropomyosin can differ significantly from that in the original model. Because resolution was insufficient to assign tropomyosin side-chains, the interacting-face could not be unambiguously determined. Here, we use structural analysis and energy landscapes to further examine the proposed models. The observed bend in seven crystal structures of tropomyosin is much closer in direction and extent to the original model than to the new model. Additionally, we computed the interaction map for repositioning tropomyosin over the F-actin surface, but now extended over a much larger surface than previously (using the original interacting-face). This map shows two energy minima— one corresponding to the “blocked-state” as in the original model, and the other related by a simple 24 Å translation of tropomyosin parallel to the F-actin axis. The tropomyosinactin complex defined by the second minimum fits perfectly into the recent cryo-EM density, without requiring any change in the interacting-face. Together, these data suggest that movement of tropomyosin between regulatory states does not require interacting-face rotation. Further, they imply that thin filament assembly may involve an interplay between initially seeded tropomyosin molecules growing from distinct binding-site regions on actin.

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

“…Additionally, we will run new MD simulations to explore alternative Tpm positions as revealed by recent cryo-EM (Sousa et al 2013;von der Ecken et al 2015) and computational studies (Rynkiewicz et al 2015), which were not explored by the current simulation due to limited computing resource.…”

The propensity for tropomyosin twisting in the presence and absence of F-actin

“…This model of tropomyosin displays canonical super-helical coiled-coil shape needed for binding F-actin and has been optimized to maximize potential electrostatic interactions between the two structures. The Li model is a refined version of a canonical structure originally developed by Holmes and Lorenz [32], but now with residue orientations and amino acid side-chain placement specified by their positions in crystal structures [15,33], in which the resulting structure was subjected to extensive energy minimization and MD analysis [33]. Experimental verification of the model is provided by the near perfect docking into reconstructed EM volumes of actin-tropomyosin generated by Li et al .…”

“…Here the model is shaped to lie at a 39 Å radius relative to the central axis of F-actin, as in cryo-EM structures of actin-tropomyosin [35,36] and as critically defined by diffraction of oriented actin-tropomyosin gels [32]. The model shows that tropomyosin is primarily linked to actin by numerous weak electrostatic interactions that provide about 8-fold greater binding energy than even weaker van der Waals forces [30,33]. While the Li et al .…”

“…Energy landscape measurement provides a convenient way to assess the likely energy costs of sliding (or rolling) tropomyosin around F-actin [30,32,33,38]. By translating the canonical tropomyosin model over the surface of F-actin in silico , we previously showed that interaction energetics of the actin-tropomyosin complex are defined by two broad energy minima [33].…”

“…Pseudo-rotated tropomyosins were generated computationally [32] and minimized [33] using previously published methods [15,30]. The program TWISTER [34] was run on MD frames and the resulting coiled-coil twist (phase) angles were compared to those obtained on the Li et al .…”