Structure and Dynamics of the Flexible Cardiac Troponin T Linker Domain in a Fully Reconstituted Thin Filament

Abstract

The structural analysis of large protein complexes has been greatly enhanced through the application of electron microscopy techniques. One such multiprotein complex, the cardiac thin filament (cTF), has cyclic interactions with thick filament proteins to drive contraction of the heart that has recently been the subject of such studies. As important as these studies are, they provide limited or no information on highly flexible regions that in isolation would be characterized as inherently disordered. One such region is the extended cardiac troponin T (cTnT) linker between the regions of cTnT which have been labeled TNT1 and TNT2. It comprises a hinge region (residues 158–166) and a highly flexible region (residues 167–203). Critically, this region modulates the troponin/tropomyosin complex’s position across the actin filament. Thus, the cTnT linker structure and dynamics are central to the regulation of the function of cardiac muscles, but up to now, it was ill-understood. To establish the cTnT linker structure, we coupled an atomistic computational cTF model with time-resolved fluorescence resonance energy transfer measurements in both ±Ca2+ conditions utilizing fully reconstituted cTFs. We mapped the cTnT linker’s positioning across the actin filament, and by coupling the experimental results to computation, we found mean structures and ranges of motion of this part of the complex. With this new insight, we can now address cTnT linker structural dynamics in both myofilament activation and disease.

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

“…5B to D, red and blue atoms, respectively) that link it to both C-terminal and N-terminal parts of Tm that form the overlap region. TnT1 also plays a role in the cooperativity between the two strands of Tm ( 6 , 46 ), since its interaction with the junction region on the opposite strand of the TF provides a physical link between the two TF stands via TnT1 linker region ( 47 ).…”

Myofilament-associated proteins with intrinsic disorder (MAPIDs) and their resolution by computational modeling

“…TnT has two IDR regions. The first is a ∼50 residue linker (approximately residues R158–Q203) between two structured motifs ( Deranek et al, 2022 ). Cross-linking mass spectroscopy (MS) shows that the binding of TnT’s intrinsically disordered C-terminus to TnC contributes to force generation in the myofilament ( Johnston et al, 2019 ).…”

“…Cross-linking mass spectroscopy (MS) shows that the binding of TnT’s intrinsically disordered C-terminus to TnC contributes to force generation in the myofilament ( Johnston et al, 2019 ). Using Foerster resonance energy transfer (FRET) and molecular dynamics (MD) simulations, the linker’s conformational ensembles on the full cardiac thin filament have been elucidated ( Deranek et al, 2022 ). The second region is the C-terminus of TnT, which has been predicted to be an IDR ( Na et al, 2016 ), and confirmed by our PONDR results in Fig.…”

“…Another example revealed TnT’s flexible linker conformations on the full cardiac thin filament. The time-resolved FRET data measured from labels in the IDR linker were then used as constraints to bias MD sampling of the TnT structure ( Deranek et al, 2022 ). Fluorescence correlation spectroscopy is another technique that can be used to probe conformational changes occurring over 20–300 ns ( Lee et al, 2015 ), but applications to myofilament proteins appear to be limited to determining protein concentrations in vivo, such as by Duggal et al (2017) .…”

“…The fluorescence techniques introduced in section ‘ Experimental techniques ’ lend themselves to probing the binding between IDRs and their targets ( Lee et al, 2015 ). As one example, time-resolved FRET has been used in tandem with MD simulations to identify functionally important conformations of TnT’s IDR linker when bound to the thin filament ( Deranek et al, 2022 ). Another example monitored differences in intrinsic fluorescence as a probe for conformational changes during TnT/TnC binding; this study also revealed that a DCM-associated mutation in TnC enhanced their binding ( Johnston et al, 2019 ).…”