Structural Changes Induced by the Binding of the Calcium Desensitizer W7 to Cardiac Troponin

Abstract

Compounds that directly modulate the affinity of the thin filament calcium regulatory proteins in cardiac muscle have potential for treating heart disease. A recent "proof of concept" study showed that the desensitizer W7 can correct hyper-calcium-sensitive sarcomeres from RCM R193H inhibitory subunit troponin I (cTnI) transgenic mice. We have determined the high-resolution nuclear magnetic resonance solution structure of W7 bound to the regulatory domain of calcium binding subunit troponin C (cNTnC)−cTnI cChimera designed to represent the key aspects of the cTnC−cTnI interface. The structure shows that W7 does not perturb the overall structure of the cTnC−cTnI interface, with the helical structure and position of the cTnI switch region remaining intact upon W7 binding. The naphthalene ring of W7 sits in the hydrophobic pocket created by the cNTnC−cTnI switch peptide interface, while the positively charged amine tail extends into the solvent. The positively charged tail of W7 is in the proximity of Arg147 of the cTnI switch region, supporting the suggestion that electrostatic repulsion is an aspect underlying the mechanism of desensitization. Ser84 (replacing the unique Cys84 in cTnC reported to make a reversible covalent bond with levosimendan) also contacts W7.

X-ray structure of a human cardiac muscle troponin C/troponin I chimera in two crystal forms

“…A second clone was prepared that included a C-terminal histidine tag (Table 1) in order to precisely mimic the protein used in the reported NMR structure (Cai et al, 2018), expressed and lysed as described above. Instead of loading onto a Q column, the protein was loaded onto a 5 ml HisTrap FF (GE Healthcare) column equilibrated with buffer A (20 mM Tris pH 8, 300 mM NaCl, 1 mM CaCl 2 , 1 mM DTT, 20 mM imidazole).…”

“…The reduced anisotropic structure-factor data file was 93.4% complete to 1.81 A ˚resolution (Table 2). Molecular-replacement models were prepared based on the reported NMR structures of the cTnC-cTnI chimera, including Protein Data Bank (PDB) entries 5vln (Cai et al, 2016) and 6mv3 (Cai et al, 2018). Additional models were made based on the NMR structures of the cTnC-cTnI complex (PDB entry 2mkp; Robertson et al, 2014) as well as the corresponding region of the crystal structure of the troponin core domain (PDB entry 1j1e; Takeda et al, 2003).…”

“…In NMR structures of the cTnC-cTnI chimera bound to ligands such as bepridil (Wang et al, 2002) and W7 (Cai et al, 2018), the binding site on the hydrophobic groove of cTnC includes Leu41, Leu48, Met60 and Met80. In the NMR structures, these residues of the hydrophobic core are exposed and movement of the cTnI peptide is not required for the ligand to bind.…”

“…The use of a chimera greatly increases the effective concentration and promotes the stabilization of the cTnI switch region. Using such a chimeric protein, a number of NMR structures have been determined (Pineda-Sanabria et al, 2014;Cai et al, 2016Cai et al, , 2018. These structures show an interaction between cTnC and cTnI similar to that seen in the native structure, with the cTnI portion forming an approximately three-turn helix running antiparallel to the cTnC portion of the chimera.…”

Small Molecule RPI-194 Stabilizes Activated Troponin to Increase the Calcium Sensitivity of Striated Muscle Contraction

“…We, therefore, designed and screened compounds for binding to the cTn complex using a unique cNTnC-cTnI chimeric construct, which we named “gChimera” ( Cai et al, 2018 ). We synthesized a novel small molecule cardiac troponin modulator, RPI-194, and measured its binding to both gChimera and to the isolated cNTnC domain, as well as its activity in skinned cardiac muscle trabeculae, individual cardiomyocytes, and isolated perfused working mouse hearts.…”