High-resolution cryo-EM structure of the junction region of the native cardiac thin filament in relaxed state

Abstract

Cardiac contraction depends on molecular interactions among sarcomeric proteins coordinated by the rising and falling intracellular Ca²⁺ levels. Cardiac thin filament (cTF) consists of two strands composed of actin, tropomyosin (Tm), and equally spaced troponin (Tn) complexes forming regulatory units. Tn binds Ca²⁺ to move Tm strand away from myosin-binding sites on actin to enable actomyosin cross-bridges required for force generation. The Tn complex has three subunits-Ca²⁺-binding TnC, inhibitory TnI, and Tm-binding TnT. Tm strand is comprised of adjacent Tm molecules that overlap "head-to-tail" along the actin filament. The N-terminus of TnT (e.g., TnT1) binds to the Tm overlap region to form the cTF junction region-the region that connects adjacent regulatory units and confers to cTF internal cooperativity. Numerous studies have predicted interactions among actin, Tm, and TnT1 within the junction region, although a direct structural description of the cTF junction region awaited completion. Here, we report a 3.8 Å resolution cryo-EM structure of the native cTF junction region at relaxing (pCa 8) Ca²⁺ conditions. We provide novel insights into the "head-to-tail" interactions between adjacent Tm molecules and interactions between the Tm junction with F-actin. We demonstrate how TnT1 stabilizes the Tm overlap region via its interactions with the Tm C- and N-termini and actin. Our data show that TnT1 works as a joint that anchors the Tm overlap region to actin, which stabilizes the relaxed state of the cTF. Our structure provides insight into the molecular basis of cardiac diseases caused by missense mutations in TnT1.

Fig. 1.

The structural details of the cardiac TF and 3D reconstruction of its junction region at low Ca²⁺ (pCa 8). (A) The model of the cTF. The backbone of the cTF is comprised of actin (tan ribbons). Components of the Tn complex, which include TnC, TnI, and TnT are shown as blue, red, and cyan ribbons, respectively. Two consecutive Tm molecules are shown as green and gold ribbons. TnT is a part of both Tn core (black square bracket) and junction region (magenta square bracket). The two unequal Tn complexes are marked as upper (U-Tn) and lower (L-Tn). (B) The 3D reconstruction of the junction region is shown at 3.8 Å resolution using colored codes from (A). The barbed end of actin is at the bottom.

Fig. 2.

The interstrand interactions of the α-Tm N’ and C’ termini of the cardiac native TF. (A) Diagram of the Tm seven-residue heptad. Residues in a/a’ (blue double-headed arrow) and d/d’ (blue double-headed arrow) heptad positions form hydrophobic core interactions (B and C, blue residues). Canonical salt bridges are formed by residues in e/g’ (magenta double-headed arrow) and e’/g (magenta double-headed arrow), while noncanonical salt bridges have been observed between a and g’ (cyan double-headed arrow). (B) Interactions between the Tm strands in the C-terminus (green ribbons) are comprised of hydrophobic interactions (blue atoms) and salt bridges (magenta atoms). The C-terminus begins to splay at Q263 (red atoms) and is separated further by the large side chains of Y267 (red atoms). (C) Interactions between the Tm stands in the N-terminus (gold ribbons) involve hydrophobic interactions (blue atoms) and salt bridges (magenta atoms). Two noncanonical salts bridges are shown in cyan.

Fig. 3.

The proposed intermolecular interactions of the α-Tm overlap region of the cardiac native TF. The Tm overlap region between the C-terminus (green ribbons) and the N-terminus (gold ribbons) of the adjacent Tm molecule spans three actin subunits (tan ribbons). The black box outlines the detailed part of the model. This part docked into the corresponding density map region is shown in Supplementary Information Appendix, Fig. S5A. The Tm C-terminal apolar residues are shown as magenta atoms, while hydrophobic residues located in the Tm N-terminus are shown as blue atoms. Charged residues that form ionic bonds are shown in red, while the two lysines (e.g., K7 and K12) that contribute to hydrophobic interactions are marked in cyan. Interaction between I284 and apolar part of K12 is marked with black arrow. The salt bridge between Tm D280 and K5 is marked with red arrow. Proposed interactions between C-terminus residues and N-terminus residues that stabilize the Tm overlap region are discussed in the text. The barbed end of actin is at the bottom.

Fig. 4.

Proposed interactions of the Tm coiled-coil with actin. Actin is shown in tan, while the Tm periods are shown in different colors. C-terminal periods 6 and 7 presented in the model are in dark and light green, respectively. N-terminal periods 1 and 2 are shown in gold and dark red, respectively. Actin and Tm residues involved in the ionic interactions (colored spheres) are discussed in the text. These residues with the corresponding regions of the density map are shown in Supplementary Information Appendix, Fig. S6. The barbed end of actin is at the bottom.

Fig. 5.

Proposed interactions of TnT1 with Tm and actin. (A) Atomic model of the cTF junction region is shown having actin in tan, Tm’s C-terminus in green, Tm’s N-terminus in gold, and TnT1 in cyan ribbon. The two views of the same model are related by 90° azimuthal rotation. (B to G) Detailed views of TnT1 interface with Tm (B to E) and actin (F and G) are shown in colored boxes, which are related to the cTF junction region model shown in (A). TnT1 and Tm residues that form salt bridges are shown as red atoms, while TnT1 and Tm residues involved in hydrophobic interactions are shown as blue atoms. TnT1 and actin residues that form salt bridges are depicted as magenta atoms. TnT1 and actin residues that form a hydrophobic cluster under subdomain 1 of actin are outlined as purple atoms. TnT1 residues involved in preservation of the geometry of the TnT1 loop that connects the two actin-binding regions on TnT1 (i.e., ionic magenta and hydrophobic purple) are depicted as black atoms. Red arrows point to residue pairs that form salt bridges. Residues that have pathological variants linked to cardiomyopathies are marked with an asterisk. (H) TnT1 porcine and their corresponding pathogenic variants in humans are tabulated using the same color codes used in (B to G). Details of interactions are discussed in the text. The residues involved in TnT1 interactions with Tm and actin are shown along with the corresponding regions of the density map in Supplementary Information Appendix, Fig. S8.