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

Abstract

Background: Cardiac troponin I (cTnI) has two flexible tails that control the cardiac cycle. The C-terminal tail, cTnI_135-209, binds actin to shut off cardiac muscle contraction, whereas the competing calcium-dependent binding of the switch region, cTnI_146-158, by cardiac troponin C (cTnC) triggers contraction. The N-terminal tail, cTnI_1-37, 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.

Methods: Soluble fragments of cTnI containing its N- and C-terminal tails, cTnI_1-77 and cTnI_135-209, 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.

Results: cTnI_135-209 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.

Conclusions: 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.

General significance: 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.

Keywords: Calpain; Intrinsically disordered protein; Ischemia-reperfusion injury; Mass spectrometry; Matrix metalloproteinase-2; Myocardial infarction; Myocardial stunning; Nuclear magnetic resonance (NMR) spectroscopy.