Abstract
The hypertrophic cardiomyopathy-associated mutant D145E, in cardiac troponin C (cTnC) C-domain, causes generalised instability at multiple sites in the isolated protein. As a result, structure and function of the mutant are more susceptible to higher temperatures. Above 25 °C there are large, progressive increases in N-domain Ca2+-binding affinity for D145E but only small changes for the wild-type protein. NMR-derived backbone amide temperature coefficients for many residues show a sharp transition above 30–40 °C, indicating a temperature-dependent conformational change that is most prominent around the mutated EF-hand IV, as well as throughout the C-domain. Smaller, isolated changes occur in the N-domain. Cardiac skinned fibres reconstituted with D145E are more sensitive to Ca2+ than fibres reconstituted with wild-type, and this defect is amplified near body-temperature. We speculate that the D145E mutation destabilises the native conformation of EF-hand IV, leading to a transient unfolding and dissociation of helix H that becomes more prominent at higher temperatures. This creates exposed hydrophobic surfaces that may be capable of binding unnaturally to a variety of targets, possibly including the N-domain of cTnC when it is in its open Ca2+-saturated state. This would constitute a potential route for propagating signals from one end of TnC to the other.
Highlights
Cardiac troponin C controls key events of systole and diastole through its ability to bind and release Ca2+, and changes in its Ca2+ affinity can promote a systolic or diastolic dysfunction that leads to a chronic problem, typically followed by remodeling of the ventricular wall. cardiac troponin C (cTnC) has two globular domains connected by a long flexible linker and three EF-hand sites for Ca2+ binding; when the TnI switch peptide binds, the B and C helices in the cTnC N-domain swing out away from the others to accommodate it[1, 2]
Since Ca2+ binding to cTnC leads to global structural changes[13, 14], we postulated that stability and folding might be disrupted, and we sought to correlate the stability of the human cTnC (HcTnC) D145E with its physiological function in skinned fibres
Several backbone assignments are available for WT cardiac troponin C in the Biological Magnetic Resonance Data Bank[21, 22], none matches exactly the entire primary sequence and buffer conditions used in this work
Summary
Cardiac troponin C (cTnC) controls key events of systole and diastole through its ability to bind and release Ca2+, and changes in its Ca2+ affinity can promote a systolic or diastolic dysfunction that leads to a chronic problem, typically followed by remodeling of the ventricular wall. cTnC has two globular domains connected by a long flexible linker and three EF-hand sites for Ca2+ binding; when the TnI switch peptide binds, the B and C helices in the cTnC N-domain swing out away from the others to accommodate it[1, 2]. Its capacity for causing diastolic dysfunction has been attributed to its effect at the N-domain site II, where it increases the affinity for Ca2+ and delays cardiac muscle relaxation[8, 10] This mutation causes minor changes in certain structural parameters, including the α-helical content[11]. We have looked for evidence of changes in the internal dynamics of the N- and C-domains of the mutants[18] Since in these experiments temperatures near the physiological range promoted substantial changes in HcTnC D145E structure that were not seen in the WT protein, we investigated the effect of temperature on the function of HcTnC D145E. A second aim was to evaluate the temperature dependence of stability and function for the HcTnC D145E mutant incorporated into skinned cardiac myofibrils and compare it with the WT protein using temperatures in the near-physiological range
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