Three-Dimensional Organization of Troponin on Cardiac Muscle Thin Filaments in the Relaxed State

Abstract

Muscle contraction is regulated by troponin-tropomyosin, which blocks and unblocks myosin binding sites on actin. To elucidate this regulatory mechanism, the three-dimensional organization of troponin and tropomyosin on the thin filament must be determined. Although tropomyosin is well defined in electron microscopy helical reconstructions of thin filaments, troponin density is mostly lost. Here, we determined troponin organization on native relaxed cardiac muscle thin filaments by applying single particle reconstruction procedures to negatively stained specimens. Multiple reference models led to the same final structure, indicating absence of model bias in the procedure. The new reconstructions clearly showed F-actin, tropomyosin, and troponin densities. At the 25 Å resolution achieved, troponin was considerably better defined than in previous reconstructions. The troponin density closely resembled the shape of troponin crystallographic structures, facilitating detailed interpretation of the electron microscopy density map. The orientation of troponin-T and the troponin core domain established troponin polarity. Density attributable to the troponin-I mobile regulatory domain was positioned where it could hold tropomyosin in its blocking position on actin, thus suggesting the underlying structural basis of thin filament regulation. Our previous understanding of thin filament regulation had been limited to known movements of tropomyosin that sterically block and unblock myosin binding sites on actin. We now show how troponin, the Ca(2+) sensor, may control these movements, ultimately determining whether muscle contracts or relaxes.

Regulatory domain of troponin moves dynamically during activation of cardiac muscle

“…4 A and E), which is broadly consistent with a previous model for the skeletal muscle thin filament (Model 2 of Knowles et al) [12] and the recently published model of cardiac thin filament in the relaxed state using a single-particle reconstruction technique [23]. It is also in general agreement with previous models of the thin filament based on EM and FRET data in which the arrowhead-shaped IT complex points towards the barbed end of the actin filament [10,24,25], but is in contrast to an earlier model based on single-particle analysis of reconstructed thin filament [11].…”

Thin Filament Structure and the Steric Blocking Model

“…The reconstructions revealed that the fragment competes with tropomyosin for a common site and fixes tropomyosin in the B-state position seen in low-Ca maps. Yang et al (229) later confirmed these results by reconstructing whole troponin on thin filaments. The work therefore directly connected troponin action to steric regulation.…”

“…While noise reduction is advantageous, helical reconstruction is very tedious and cannot be easily used to categorize variants within a class of filaments or, as mentioned, to reconstruct nonhelically arranged thin-filament elements. To determine troponin structure, for example, different reconstruction methods are needed to recover troponin density contributions on actin-tropomyosin (174,175,229).…”

Tropomodulins and Leiomodins: Actin Pointed End Caps and Nucleators in Muscles

“…The structure of the two TM-binding sites (green) is unknown, and a tentative model was generated based on secondary structure prediction and energy minimization (5). TM is shown in the blocked state, which it assumes when bound to the filament with Ca2þ-free troponin (53,111). Of note, this model is consistent with TM's ability to explore all three states on the filament (blocked, closed, and open) (53,54), without generating steric clashes with Tmod at the pointed end.…”