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 cTnI147–163 or bepridil. This raises the question of whether cTnI147–163 and bepridil compete for cNTnC·Ca2+. In this work, we used two-dimensional1H,15N-heteronuclear single quantum coherence (HSQC) NMR spectroscopy to examine the binding of bepridil to cNTnC·Ca2+ in the absence and presence of cTnI147–163 and of cTnI147–163 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 cTnI147–163 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+·cTnI147–163·bepridil ternary complex. The structure reveals a binding site for cTnI147–163 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 cTnI147–163 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.