Effects of Calcium Binding and the Hypertrophic Cardiomyopathy A8V Mutation on the Dynamic Equilibrium between Closed and Open Conformations of the Regulatory N-Domain of Isolated Cardiac Troponin C

Abstract

Troponin C (TnC) is the calcium-binding subunit of the troponin complex responsible for initiating striated muscle contraction in response to calcium influx. In the skeletal TnC isoform, calcium binding induces a structural change in the regulatory N-domain of TnC that involves a transition from a closed to open structural state and accompanying exposure of a large hydrophobic patch for troponin I (TnI) to subsequently bind. However, little is understood about how calcium primes the N-domain of the cardiac isoform (cTnC) for interaction with the TnI subunit as the open conformation of the regulatory domain of cTnC has been observed only in the presence of bound TnI. Here we use paramagnetic relaxation enhancement (PRE) to characterize the closed to open transition of isolated cTnC in solution, a process that cannot be observed by traditional nuclear magnetic resonance methods. Our PRE data from four spin-labeled monocysteine constructs of isolated cTnC reveal that calcium binding triggers movement of the N-domain helices toward an open state. Fitting of the PRE data to a closed to open transition model reveals the presence of a small population of cTnC molecules in the absence of calcium that possess an open conformation, the level of which increases substantially upon Ca(2+) binding. These data support a model in which calcium binding creates a dynamic equilibrium between the closed and open structural states to prime cTnC for interaction with its target peptide. We also used PRE data to assess the structural effects of a familial hypertrophic cardiomyopathy point mutation located within the N-domain of cTnC (A8V). The PRE data show that the Ca(2+) switch mechanism is perturbed by the A8V mutation, resulting in a more open N-domain conformation in both the apo and holo states.

Ca2+-Induced PRE-NMR Changes in the Troponin Complex Reveal the Possessive Nature of the Cardiac Isoform for Its Regulatory Switch

“…Our results support a model of the cardiac isoform where the cTnI switch peptide is clearly released from the N-lobe of cTnC in the low Ca 2+ state; however, upon release, it remains in close vicinity to cTnC. We speculate that the close proximity of cTnI to cTnC is favourable for increasing the frequency of collisions between cTnC and the switch region, counteracting the reduced probability of collisions due to the incomplete opening of the N-lobe of cTnC in the cardiac isoform [11].…”

“…To prepare calcium free samples (−Ca 2+ ), Ca 2+ was removed by dialysis against 0.1 M EGTA for 2 h at 4°C followed by exhaustive dialysis into decalcified NMR buffer (treated with Chelex-100 resin, Bio-Rad) with 5 mM MgCl 2 , as previously described [11].…”

“…Separating these two regions of TnI is an amphipathic helix termed the ‘switch region’ (cardiac residues 150–159). The current model for muscle regulation posits that Ca 2+ binding promotes the opening of the regulatory N-lobe of TnC to reveal a hydrophobic pocket which allows for binding of the TnI switch region [11], [12]. It is this interaction of the switch region with TnC that is also required to stabilise the ‘open’ conformation of the N-lobe of the cardiac isoform of TnC [13].…”

Regulatory domain of troponin moves dynamically during activation of cardiac muscle

“…Such a dynamic movement of the regulatory head may increase the probability of it interacting with the TnI switch peptide when Ca 2+ is bound, and thus compensate for the initial small structural change of cTnC upon Ca 2+ binding alone [17,18]. The broad orientational distribution of NTnC in the relaxed state may be an indication that some molecules are in the open conformatiom state even in the absence of Ca 2+ [27] and may partly contribute to the lower density observed in electron microscopy reconstructions [23]. Thus the present finding suggests conformational dynamics of Tn crucial to its function, but difficult to detect experimentally.…”

Allosteric Transmission Along a Loosely Structured Backbone Allows a Cardiac Troponin C Mutant to Function with only One Ca 2+ ion

“…The single-residue substitution at position 8 in the N-helix of cTnC (A8V) confers an increase of the ␣-helical content (11). Although the overall 1 H- 15 N HSQC spectrum appears similar to that of the wild-type (WT) form, this mutation seems to increase the population of the N-domain open conformation (12). In the case of A31S, the subject carrying this mutation is prone to develop ventricular fibrillation.…”