Structure of the regulatory N-domain of human cardiac troponin C in complex with human cardiac troponin I147-163 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 H-1,N-15-heteronuclear single quantum coherence (HSQC) NAIR 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 cTnI147-163 bind cNTnC.Ca2+ simultaneously but with negative cooperativity. The affinity of cTnI(147-163) for cNTnC.Ca2+ is reduced similar to 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.