C-Terminal Basic Region of Troponin T Alters the Ca²⁺-Dependent Changes in Troponin I Interactions

Abstract

The C-terminal 14–16 residues of human troponin T are required for full inactivation, and they prevent full activation at saturating Ca2+. Basic residues within that C-terminal region of TnT are essential for its function, but the mechanism of action is unknown. That region of TnT is natively disordered and does not appear in reconstructions of the troponin structure. We used Förster resonance energy transfer to determine if the C-terminal basic region of TnT alters transitions of TnI or if it operates independently. We also examined Ca2+-dependent changes in the C-terminal region of TnT itself. Probes on TnI-143 (inhibitory region) and TnI-159 (switch region) moved away from sites on actin and tropomyosin and toward TnC-84 at high Ca2+. Ca2+ also displaced C-terminal TnT from actin–tropomyosin but without movement toward TnC. Deletion of C-terminal TnT produced changes in TnI-143 like those effected by Ca2+, but effects on TnI-159 were muted; there was no effect on the distance of the switch region to TnC-84. Substituting Ala for basic residues within C-terminal TnT displaced C-terminal TnT from actin–tropomyosin. The results suggest that C-terminal TnT stabilizes tropomyosin in the inactive position on actin. Removal of basic residues from C-terminal TnT produced a Ca2+-like state except that the switch region of TnI was not bound to TnC. Addition of Ca2+ caused more extreme displacement from actin–tropomyosin as the active state became more fully occupied as in the case of wild-type TnT in the presence of both Ca2+ and bound rigor myosin S1.

Hypertrophic Cardiomyopathy Mutations of Troponin Reveal Details of Striated Muscle Regulation

“…We predict from these observations that the CB region of TnT stabilizes the binding of the switch and C-terminal regions of TnI to the actin filament. That hypothesis is supported by FRET measurements between these regions of TnI and both actin-374 and tropomyosin-190 ( Zhu et al, 2022 ).…”

“…FRET measurements showed that in forming the inactive-Ca 2+ -free state from the inactive-Ca 2+ -bound state, CB residues 275 and 289 approach tropomyosin-190 ( Johnson et al, 2019 ; Zhu et al, 2022 ). Residue 275 was closer to tropomyosin-190 than residue 289 of TnT.…”

“…Further FRET studies showed that Ca 2+ binding to TnC caused the CB region of TnT to move away from actin as well as from tropomyosin ( Zhu et al, 2022 ). That is, there appears to be a coordinated movement of regions of TnT and TnI away from actin-tropomyosin upon Ca 2+ binding.…”

“…The CB region of TnT might also bind near the N-lobe of TnC in the active state ( Johnston et al, 2019 ). However, FRET measurements show that the CB region of TnT remains far from TNC at both low and high free Ca 2+ levels ( Zhu et al, 2022 ). Because of the potential importance of the CB region in regulation, this discrepancy must be resolved.…”

Negative Charges Introduced Near the IT Helix of Cardiac Troponin T Stabilize the Active State of Actin Filaments

“…There are indications, however, that the CB region of TnT is bound to tropomyosin in the virtual absence of Ca 2+ . Probes placed on either residue 275 or 289 of TnT moved away from Cys 190 of tropomyosin , and away from Cys 374 of actin upon the addition of Ca 2+ . Removal of the positive charges from the CB region of TnT mimicked the effect of adding Ca 2+ but the further addition of Ca 2+ produced an additional displacement .…”