Versatile Cardiac Troponin Chimera for Muscle Protein Structural Biology and Drug Discovery

Abstract

Investigation of the molecular interactions within and between subunits of the heterotrimeric troponin complex, and with other proteins in the sarcomere, has revealed salient structural elements involved in regulation of muscle contraction. The discovery of new cardiotonic drugs and structural studies utilizing intact troponin, or regulatory complexes formed between the key regions identified in troponin C and troponin I, face intrinsic and technical difficulties associated with weak protein-protein interactions and with solubility, aggregation, stability of the overall architecture, isotope labeling, and size, respectively. We have designed and characterized a chimeric troponin C-troponin I hybrid protein with a cleavable linker that is useful for producing isotopically labeled troponin peptides, stabilizes their interaction, and has proven to be a faithful representation of the original complex in the systolic state, but lacking its disadvantages, making it particularly suitable for drug screening and structural studies.

Myofilament Calcium Sensitivity: Consequences of the Effective Concentration of Troponin I

“…If we include more residues, 134–155, we estimate a lower limit of ~3 mM. This is very much in line with previous estimates of the effective concentration of a chimeric TnC-TnI protein (Pineda-Sanabria et al, 2014). …”

“…If we then place a single switch peptide into this volume we can calculate what the “effective concentration” of this peptide would be for a TnC that shares this volume space, ~3000 μM. This calculated value is similar to that estimated for another TnC-TnI chimeric protein by NMR (Hwang et al, 2014; Pineda-Sanabria et al, 2014). If we also assume that there are regions of this volume that TnC does not share, then it is possible to potentially trap, or at least temporarily restrict, TnI away from TnC, drastically plummeting the effective concentration of TnI that TnC “observes.” Such an occurrence is not difficult to imagine when the Tn complex is docked onto the thin filament, since the C-terminal domain of TnI can bind both TnC and actin (Tripet et al, 1997; Figure 3B).…”

“…One such behavior, effective concentration, emerges when reactants are restricted to interact in very confined spaces, as occurs when two reactants are physically tethered together (Van Valen et al, 2009). TnC and TnI can be both artificially tethered in chimeras (Tiroli et al, 2005; Pineda-Sanabria et al, 2014) and more naturally in the Tn complex (Pineda-Sanabria et al, 2014; Davis J. P. et al, 2016). Due to being tethered (Van Valen et al, 2009), TnC has the potential to experience extremely high effective concentrations of TnI to extremely low (in times when TnI mobility becomes restricted).…”

Structures reveal details of small molecule binding to cardiac troponin

“…Hybrid proteins have been used to simplify systems with complex protein–protein interactions [19,20], including both skeletal and cardiac muscle isoforms of troponin[21,22]. Since dfbp-o and other compounds are known to bind to the interface between NTnC and TnI switch peptide[23], we developed a cardiac troponin C-troponin I chimera, cChimera, in which the switch region of cTnI is fused to the C terminus of cNTnC, building upon the previous design cNTnC-linker-switch peptide 144–173[21].…”

“…Since dfbp-o and other compounds are known to bind to the interface between NTnC and TnI switch peptide[23], we developed a cardiac troponin C-troponin I chimera, cChimera, in which the switch region of cTnI is fused to the C terminus of cNTnC, building upon the previous design cNTnC-linker-switch peptide 144–173[21]. cChimera begins with residues 1 to 90 of human cNTnC (C35S, C84S), followed by residues 136–163 of cTnI (comprising the inhibitory 136–147 and switch 147–163 regions) and ending with a C-terminal His-tag.…”