Structure and proteolytic susceptibility of the inhibitory C-terminal tail of cardiac troponin I

Cardiac troponin I (cTnI) has two flexible tails that control the cardiac cycle. The C-terminal tail, cTnI135–209, binds actin to shut off cardiac muscle contraction, whereas the competing calcium-dependent binding of the switch region, cTnI146–158, by cardiac troponin C (cTnC) triggers contraction. The N-terminal tail, cTnI1–37, regulates the calcium affinity of cTnC. cTnI is known to be susceptible to proteolytic cleavage by matrix metalloproteinase-2 (MMP-2) and calpain, two intracellular proteases implicated in ischemia-reperfusion injury. Soluble fragments of cTnI containing its N- and C-terminal tails, cTnI1–77 and cTnI135–209, were highly expressed and purified from E. coli. We performed in vitro proteolysis studies of both constructs using liquid chromatography-mass spectrometry and solution NMR studies of the C-terminal tail. cTnI135–209 is intrinsically disordered, though it contains three regions with helical propensity (including the switch region) that acquire more structure upon actin binding. We identified three precise MMP-2 cleavage sites at cTnI P17-I18, A156-L157, and G199-M200. In contrast, calpain-2 has numerous cleavage sites throughout Y25-T30 and A152-A160. The critical cTnI switch region is targeted by both proteases. Both N-terminal and C-terminal tails of cTnI are susceptible to cleavage by MMP-2 and calpain-2. Binding to cTnC or actin confers some protection to proteolysis, which can be understood in terms of their interactions as probed by NMR studies. cTnI is an important marker of intracellular proteolysis in cardiomyocytes, given its many protease-specific cut sites, high natural abundance, indispensable functional role, and clinical use as gold standard biomarker of myocardial injury. •The disordered C-terminal tail of cardiac troponin I (residues 135–209) alternates between actin and cardiac troponin C to control the cardiac cycle•The C-terminal tail becomes partially structured upon binding actin•Both N- and C-terminal tails are susceptible to proteolysis by intracellular proteases MMP-2 and calpain-2•Both MMP-2 and calpain-2 cleave the critical switch region of cardiac troponin I, though it is protected when bound to actin or troponin C