Calcium‐regulated conformational change in the C‐terminal end segment of troponin I and its binding to tropomyosin

Abstract

Summary The troponin complex plays an essential role in the thin filament regulation of striated muscle contraction. Of the three subunits of troponin, troponin I (TnI) is the actomyosin ATPase inhibitory subunit and its effect is released upon Ca2+-binding to troponin C. The exon 8-encoded COOH-terminal end segment represented by the last 24 amino acids of cardiac TnI is highly conserved and critical to the inhibitory function of troponin. Here we investigated the function and calcium regulation of the COOH-terminal end segment of TnI. A TnI model molecule was labeled with Alexa fluor 532 at a Cys engineered at the COOH-terminal end and used to reconstitute tertiary troponin complex. A Ca2+-regulated conformational change in the COOH terminus of TnI was shown by a sigmoid-shape fluorescence intensity titration curve similar to that of the circular dichroism calcium titration curve of troponin C. Such corresponding Ca2+-responses are consistent with the function of troponin as a coordinated molecular switch. Reconstituted troponin complex containing a mini-troponin T lacking its two tropomyosin-binding sites showed a saturable binding to tropomyosin at pCa 9 but not at pCa 4. This Ca2+-regulated binding was diminished when the COOH-terminal 19 amino acids of cardiac TnI were removed. These results provided novel evidence for suggesting that the COOH-terminal end segment of TnI participates in the Ca2+-regulation of muscle thin filament through an interaction with tropomyosin.

Functional significance of C-terminal mobile domain of cardiac troponin I

“…Metskas et al [82] have suggested in the closed state of Tm, cTnI-MD around residues 193–210 stays bound to actin and retains a conformation similar to the blocked state while it is dynamic and unbound between residues 161–192 [82]. In this regard, a previous study by Zhang et al suggested that the end segment of the C-terminus of cTnI (residues 193–210) binds to Tm [55] and TnC [56] in a Ca 2+ sensitive manner, and helps to restrict Tm movement in the relaxed condition [55]. These studies suggest that losing this segment could potentially affect the position and/or flexibility of Tm in the relaxed state promoting actomyosin binding, which agrees with our findings as well.…”

“…The cTnI-MD is intrinsically disordered, and undergoes a disordered-ordered structural transition while interacting with actin, allowing the cTnI-MD to perform its fly-casting activity to rapidly initiate the relaxation [16, 52–54]. The C-terminal end segment of cTnI-MD (residues 193–210) has been shown to interact with tropomyosin [55] and cTnC [56] in a Ca 2+ -sensitive manner and to help restrict tropomyosin position at diastole. The C-terminal deletion of cTnI, residues 193–210, is found to be associated with myocardial stunning [57, 58].…”

TNNI1, TNNI2 and TNNI3: Evolution, regulation, and protein structure–function relationships

“…Recent studies demonstrated that the last 20 amino acids of the C-terminal end segment of TnI (residues 191–210 in cTnI) encoded by exon 8 with a highly conserved sequence is a Ca 2+ -modulated allosteric structure and interacts with tropomyosin (Zhang et al, 2011; Akhter et al, 2014). While the N-terminal extension of cTnI does not have definitive interaction with other known myofilament proteins, it plays a role in modulating the conformation of other regions of the cTnI molecule (Akhter et al, 2012).…”

“…The C-terminal region of TnI binds and stabilizes tropomyosin on the actin filament in the absence of Ca 2+ to inhibit muscle contraction (Galińska et al, 2010; Zhang et al, 2011). The C-terminal end segment (192–210) is the most conserved region of the TnI polypeptide chain (Jin et al, 2001) and a Ca 2+ -modulated allosteric structure in the troponin complex (Jin et al, 2001; Zhang et al, 2011; Wang et al, 2012a).…”

“…The C-terminal end segment (192–210) is the most conserved region of the TnI polypeptide chain (Jin et al, 2001) and a Ca 2+ -modulated allosteric structure in the troponin complex (Jin et al, 2001; Zhang et al, 2011; Wang et al, 2012a). R192H and R204H mutations in the C-terminal end segment of human cTnI cause restrictive cardiomyopathy (Mogensen et al, 2003; Gambarin et al, 2008) with alterations in the conformation and function of the TnI-TnT interface and increased binding affinity of cTnI for TnT (Akhter et al, 2014).…”