Structure of the regulatory N-domain of human cardiac troponin C in complex with human cardiac troponin I_147-163 and bepridil

Cardiac troponin C (cTnC) is the Ca²⁺-dependent switch for contraction in heart muscle and a potential target for drugs in the therapy of heart failure. Ca²⁺ binding to the regulatory domain of cTnC (cNTnC) induces little structural change but sets the stage for cTnI binding. A large "closed" to "open" conformational transition occurs in the regulatory domain upon binding ctnI_147-163 or bepridil. This raises the question of whether cTnI_147-163 and bepridil compete for cNTnC·Ca²⁺. In this work, we used two-dimensional ¹H,¹⁵N-heteronuclear single quantum coherence (HSQC) NMR spectroscopy to examine the binding of bepridil to cNTnC·Ca²⁺ in the absence and presence of cTnI_147-163 and of ctnI_147-163 to cNTnC·Ca²⁺ in the absence and presence of bepridil. The results show that bepridil and ctnI_147-163 bind cNTnC·Ca²⁺ simultaneously but with negative cooperativity. The affinity of cTnI_147-163 for cNTnC·Ca²⁺ is reduced ∼3.5-fold by bepridil and vice versa. Using multinuclear and multidimensional NMR spectroscopy, we have determined the structure of the cNTnC· Ca²⁺·cTnI_147-163·bepridil ternary complex. The structure reveals a binding site for cTnI_147-163 primarily located on the A/B interhelical interface and a binding site for bepridil in the hydrophobic pocket of cNTnC·Ca²⁺. In the structure, the N terminus of the peptide clashes with part of the bepridil molecule, which explains the negative cooperativity between cTnI_147-163 and bepridil for cNTnC·Ca²⁺. This structure provides insights into the features that are important for the design of cTnC-specific cardiotonic drugs, which may be used to modulate the Ca²⁺ sensitivity of the myofilaments in heart muscle contraction.