Structure and Orientation of Troponin in the Thin Filament

Abstract

The troponin complex on the thin filament plays a crucial role in the regulation of muscle contraction. However, the precise location of troponin relative to actin and tropomyosin remains uncertain. We have developed a method of reconstructing thin filaments using single particle analysis that does not impose the helical symmetry of actin and is independent of a starting model. We present a single particle three-dimensional reconstruction of the thin filament. Atomic models of the F-actin filament were fitted into the electron density maps and troponin and tropomyosin located. The structure provides evidence that the globular head region of troponin labels the two strands of actin with a 27.5-Å axial stagger. The density attributed to troponin appears tapered with the widest point toward the barbed end. This leads us to interpret the polarity of the troponin complex in the thin filament as reversed with respect to the widely accepted model.Regulation of actin filament function is a fundamental biological process with implications ranging from cell migration to muscle contraction. Skeletal and cardiac muscle thin filaments consist of actin and the regulatory proteins troponin and tropomyosin. Contraction is initiated by release of Ca 2ϩ into the sarcomere and the consequent binding of Ca 2ϩ to regulatory sites on troponin. Troponin is believed to undergo a conformational change leading to an azimuthal movement of tropomyosin, which allows myosin heads to interact with actin, hydrolyze ATP, and generate force. The molecular basis by which troponin acts to regulate muscle contraction is only partly understood. It is essential that the structure of troponin in the thin filament at high and low Ca 2ϩ is determined to properly understand the mechanism of regulation.The basic structure of the thin filament was described by Ebashi in 1972 (1). In this structure each tropomyosin molecule covers seven actin monomers, and there is a 27.5-Å stagger between troponin molecules. The 7-Å tropomyosin structure (2), the atomic model of F-actin (3), and the troponin "core domain" (4) have recently been used to generate atomic models of the thin filament in low and high Ca 2ϩ states (5). While the position of troponin in these models was constrained by known distance measurements between filament components, the exact arrangement of the complex on the filament has not been determined a priori. Although recently published crystal structures of partial troponin complexes (4, 6) have provided valuable insights into the arrangement of the globular head or core domain, the complex in its entirety has not been crystallized.Troponin is believed to consist of a globular core domain with an extended tail (7). The globular core contains the Ca 2ϩ -binding subunit (TnC), 2 the inhibitory subunit (TnI), and the C-terminal part (residues 156 -262) of the tropomyosin-binding subunit (TnT). The extended tail consists of the N-terminal part of TnT (residues 1-155). A structural rearrangement associated with Ca 2ϩ dissociation from the troponin cor...

Thin Filament Structure and the Steric Blocking Model

“…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).…”

A Three-Dimensional FRET Analysis to Construct an Atomic Model of the Actin–Tropomyosin–Troponin Core Domain Complex on a Muscle Thin Filament

“…To date, structural models of the thin filament are primarily three-dimensional image reconstructions of electron microscopy (3D-EM) and X-ray diffraction measurements. [11][12][13][14][15] These models differ from each other on the location and orientation of Tn on an F-actin filament. Although the atomic structure of the Tn core domain has been determined, it is only part of the larger Tn molecule (∼ 60% of the mass).…”

Historical perspective on heart function: the Frank–Starling Law

“…The tropomyosin overlap region (head-to-tail) depicts interaction with near-neighbor tropomyosin dimer (dark-blue) (Greenfield et al 2006;Murakami et al 2008). The orientation of thin filament proteins is as follows: the N-terminal region of cardiac troponin T points towards the pointed end (M-band), while the core domain of the troponin complex is oriented to the barbed end (Z-disk) (Paul et al 2009). Interacting sites and structural location of actin-tropomyosin-troponin proteins were matched the best as possible in accordance with the available literature (Murakami et al 2008;Takeda et al 2003;Smillie 1982, 1983;Biesiadecki et al 2007Biesiadecki et al , 2010Morris and Lehrer 1984;Manning et al 2011;Tardiff 2011).…”

“…Myosin-S1 is depicted in solid green (light-green myosin-S1 to better understand its transition states). The orientation of thin filament proteins is: the N-terminal region of cTnT points towards the pointed end (M-band), while the core domain of the troponin complex is oriented to the barbed end (Z-disk) (Paul et al 2009). …”