Ca2+ and Myosin Cycle States Work as Allosteric Effectors of Troponin Activation

Abstract

In cardiac muscle, troponin (Tn) and tropomyosin inhibit actin and myosin interactions through the steric blocking of myosin binding to F-actin. Ca binding to Tn C modulates this inhibition. Thin filaments become activated upon Ca binding, which enables strong binding of myosin with a concomitant release of ATP hydrolysis products and level arm swinging responsible for force generation. Despite this level of description, the current cross-bridge cycle model does not fully define the structural events that take place within Tn during combinatorial myosin and Ca interventions. Here, we studied conformational changes within Tn bound to F-actin and tropomyosin by fluorescence lifetime imaging combined with Förster resonance energy transfer. Fluorescent dye molecules covalently bound to the Tn C C-lobe and Tn I C-terminal domain report Ca- and myosin-induced activation of Tn. Reconstituted thin filaments were deposited on a myosin-coated surface similar to an in vitro motility assay setup without filament sliding involved. Under all the tested conditions, Ca was responsible for the most significant changes in Tn activation. Rigor myosin activated Tn at subsaturated Ca conditions but not to the degree seen in thin filaments with Ca. ATP-γ-S did not affect Tn activation significantly; however, blebbistatin induced significant activation at subsaturating Ca levels. The relation between the extent of Tn activation and its conformational flexibility suggests that active/inactive Tn states coexist in different proportions that depend on the combination of effectors. These results satisfy an allosteric activation model of the thin filament as a function of Ca and the myosin catalytic cycle state.

Novel insights into sarcomere regulatory systems control of cardiac thin filament activation

“…Thus, these findings fit generally with conclusions from kinetic measurements of actomyosin interactions, indicating a stronger role for Ca 2+ binding than strong crossbridges in cardiac versus skeletal muscle thin filament activation. They also fit with the findings reported in dynamic measurement of thin filament activation using HS-AFM ( Matusovsky et al, 2019 ) and FRET reporters of the cTnI–cTnC interaction ( Solís et al, 2018 ; Vetter et al, 2020 ). Given that these cardiac thin filament state distributions were found in native thin filaments, further structural work should address how these distributions change in the presence of strongly bound myosin and cMyBP-C. A direct comparison of the cardiac and skeletal thin filament states using these structural techniques will be beneficial to better understand the specific regulatory differences between the two kinds of striated muscle thin filaments.…”

“…FRET measurements made in a strong binding crossbridge state induced by MgADP, which increased tension to maximum levels independent Ca 2+ , demonstrated a stabilization of the interaction of the cTnC hydrophobic patch with the cTnI switch peptide. This is consistent with FRET experiments in reconstituted, Ca 2+ -saturated thin filaments in which rigor myosin further stabilizes cTnI mobile domain proximity to cTnC relative to ATP-γ-S or blebbistatin bound to myosin ( Solís et al, 2018 ). Experiments inhibiting tension with vanadate, however, did not support an exclusive role for force-generating crossbridges in this effect on the cTnC–cTnI interaction.…”

“…Further support for the conclusion from the data of Risi et al (2017) comes from the use of FRET sensors attached to the cTnC C-lobe and cTnI C-terminal domain ( Solís et al, 2018 ). By immobilizing myosin monomers to a glass surface akin to an in vitro motility assay setup, Solís et al (2018) applied fluorescence lifetime imaging FRET to the labeled cTn bound to thin filaments in contact with myosin.…”

“…Further support for the conclusion from the data of Risi et al (2017) comes from the use of FRET sensors attached to the cTnC C-lobe and cTnI C-terminal domain ( Solís et al, 2018 ). By immobilizing myosin monomers to a glass surface akin to an in vitro motility assay setup, Solís et al (2018) applied fluorescence lifetime imaging FRET to the labeled cTn bound to thin filaments in contact with myosin. Their data demonstrated that most of the cTn activation is from Ca 2+ binding compared with the additional activating contribution of rigor myosin with a concomitant gain of the cTnI mobile domain conformational flexibility.…”

The intrinsically disordered C terminus of troponin T binds to troponin C to modulate myocardial force generation

“…Considering the abundance of basic residues at the C terminus of cTnT, this region is expected to electrostatically interact with this patch of acidic residues in cTnC. Although it has been recognized that the cardiac thin filament represents an extensive allosteric system, the molecular mechanisms are not fully understood (61)(62)(63). Our findings in this report raise an intriguing possibility that the C terminus of cTnT allosterically controls thin filament activation via modulation of the cTnI-cTnC switching mechanism.…”