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Molecular basis of calcium-sensitizing and desensitizing mutations of the human cardiac troponin C regulatory domain: a multi-scale simulation study.

ABSTRACT: Troponin C (TnC) is implicated in the initiation of myocyte contraction via binding of cytosolic Ca²? and subsequent recognition of the Troponin I switch peptide. Mutations of the cardiac TnC N-terminal regulatory domain have been shown to alter both calcium binding and myofilament force generation. We have performed molecular dynamics simulations of engineered TnC variants that increase or decrease Ca²? sensitivity, in order to understand the structural basis of their impact on TnC function. We will use the distinction for mutants that are associated with increased Ca²? affinity and for those mutants with reduced affinity. Our studies demonstrate that for GOF mutants V44Q and L48Q, the structure of the physiologically-active site II Ca²? binding site in the Ca²?-free (apo) state closely resembled the Ca²?-bound (holo) state. In contrast, site II is very labile for LOF mutants E40A and V79Q in the apo form and bears little resemblance with the holo conformation. We hypothesize that these phenomena contribute to the increased association rate, k(on), for the GOF mutants relative to LOF. Furthermore, we observe significant positive and negative positional correlations between helices in the GOF holo mutants that are not found in the LOF mutants. We anticipate these correlations may contribute either directly to Ca²? affinity or indirectly through TnI association. Our observations based on the structure and dynamics of mutant TnC provide rationale for binding trends observed in GOF and LOF mutants and will guide the development of inotropic drugs that target TnC.

SUBMITTER: Kekenes-Huskey PM

PROVIDER: S-EPMC3510055 | biostudies-literature | 2012

REPOSITORIES: biostudies-literature

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Molecular basis of calcium-sensitizing and desensitizing mutations of the human cardiac troponin C regulatory domain: a multi-scale simulation study.

Kekenes-Huskey Peter Michael PM Lindert Steffen S McCammon James Andrew JA

PLoS computational biology 20121129 11

Troponin C (TnC) is implicated in the initiation of myocyte contraction via binding of cytosolic Ca²⁺ and subsequent recognition of the Troponin I switch peptide. Mutations of the cardiac TnC N-terminal regulatory domain have been shown to alter both calcium binding and myofilament force generation. We have performed molecular dynamics simulations of engineered TnC variants that increase or decrease Ca²⁺ sensitivity, in order to understand the structural basis of their impact on TnC function. We ...[more]

PMID: 23209387

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Project description:Two cTnC variants, L57Q and I61Q, both of which are located on helix C within the N domain of cTnC, were originally reported in the skeletal muscle system [Tikunova, Davis, J. Biol. Chem. 279 (2004) 35341-35352], as the analogous L58Q and I62Q sTnC, and demonstrated a decreased Ca(2+) binding affinity. Here, we provide detailed characterization of structure-function relationships for these two cTnC variants, to determine if they behave differently in the cardiac system and as a framework for determining similarities and differences with other cTnC mutations that have been associated with DCM. We have used an integrative approach to study the structure and function of these cTnC variants both in solution and in silico, to understand how the L57Q and I61Q mutations influence Ca(2+) binding at site II, the subsequent effects on the interaction with cTnI, and the structural changes which are associated with these changes. Steady-state and stopped flow fluorescence spectroscopy confirmed that a decrease in Ca(2+) affinity for recombinant cTnC and cTn complexes containing the L57Q or I61Q variants. The L57Q variant was intermediate between WT and I61Q cTnC and also did not significantly alter cTnC-cTnI interaction in the absence of Ca(2+), but did decrease the interaction in the presence of Ca(2+). In contrast, I61Q decreased the cTnC-cTnI interaction in both the absence and presence of Ca(2+). This difference in the absence of Ca(2+) suggests a greater structural change in cNTnC may occur with the I61Q mutation than the L57Q mutation. MD simulations revealed that the decreased Ca(2+) binding induced by I61Q may result from destabilization of the Ca(2+) binding site through interruption of intra-molecular interactions when residue 61 forms new hydrogen bonds with G70 on the Ca(2+) binding loop. The experimentally observed interruption of the cTnC-cTnI interaction caused by L57Q or I61Q is due to the disruption of key hydrophobic interactions between helices B and C in cNTnC. This study provides a molecular basis of how single mutations in the C helix of cTnC can reduce Ca(2+) binding affinity and cTnC-cTnI interaction, which may provide useful insights for a better understanding of cardiomyopathies and future gene-based therapies.

Dataset Information

Molecular basis of calcium-sensitizing and desensitizing mutations of the human cardiac troponin C regulatory domain: a multi-scale simulation study.

Publications

Molecular basis of calcium-sensitizing and desensitizing mutations of the human cardiac troponin C regulatory domain: a multi-scale simulation study.

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