Weak domain stability and higher ca 2+ binding affinity contribute to allostery between the d/e linker and n-helix of cardiac troponin c
Hypertrophic cardiomyopathy (HCM) is an inherited myopathy caused by the production of anomalous sarcomeric proteins that can lead to severe cardiac dysfunction. Here, we used structural and biophysical approaches to better understand the pathogenesis of a cardiac troponin C (cTnC) C84Y mutation loc...
| Autores: | , , , , |
|---|---|
| Tipo de recurso: | artículo |
| Estado: | Versión publicada |
| Fecha de publicación: | 2018 |
| País: | Brasil |
| Institución: | Universidade Federal de Minas Gerais (UFMG) |
| Repositorio: | Repositório Institucional da UFMG |
| Idioma: | inglés |
| OAI Identifier: | oai:repositorio.ufmg.br:1843/46067 |
| Acceso en línea: | https://doi.org/10.1016/j.bpj.2017.11.2336 http://hdl.handle.net/1843/46067 https://orcid.org/0000-0002-4131-4634 https://orcid.org/0000-0002-3344-4084 https://orcid.org/0000-0001-9523-9441 https://orcid.org/0000-0001-9092-4976 https://orcid.org/0000-0002-0063-5888 |
| Access Level: | acceso abierto |
| Palabra clave: | Hypertrophic cardiomyopathy (HCM) Sarcomeric proteins Cardiac dysfunction Cardiac troponin Protein Batimento cardíaco Enzimas alostéricas Miocárdio Doenças Mutação (Biologia) |
| Sumario: | Hypertrophic cardiomyopathy (HCM) is an inherited myopathy caused by the production of anomalous sarcomeric proteins that can lead to severe cardiac dysfunction. Here, we used structural and biophysical approaches to better understand the pathogenesis of a cardiac troponin C (cTnC) C84Y mutation located in the D/E linker, first reported in a 17-year-old proband, presenting with left-ventricular hypertrophy. Despite the relevance of HCM disease, little is known concerning the function of the D/E linker and allosteric phenomena governing cTnC Ca2+ affinity. Monitored by bis-ANS fluorescence, Ca2+-titrations reveal that C84Y exhibits enhanced Ca2+-binding affinity in both domains and conformational changes compared to WT. Although WT and C84Y display distinct Ca2+-binding behaviors, the overall dimensional values and molecular envelopes generated by small-angle-X-ray scattering data remains similar. Using circular-dichroism, C84Y revealed significantly lower thermostability in non-Ca2+-bound form compared to WT. Most of our understanding of the molecular mechanisms underlying how troponin and troponin peptides switch muscle contraction “on” and “off” has been derived using experimental NMR techniques. Currently, no experimental techniques are available that allow the understanding of protein regulatory/dynamic processes at the molecular level of large, multi-domain protein complexes. To further unravel molecular changes in C84Y, three-dimensional NMR experiments were performed for backbone assignment. The largest chemical shifts were observed in N-Helix residues and at the end of D-helix and D/E linker. NMR-derived backbone amide temperature-coefficients indicate different temperature-dependent conformational changes exist between WT and C84Y Carr-Purcell-Meiboom-Gill relaxation dispersion (CPMG-RD) and R1/R2 experiments were used to probe the population and exchanging rates of C84Y compared to WT. This work sought to elucidate: main structural components underlying this pathological mutation, novel allosteric mechanisms, and the role of D/E linker in cTnC. |
|---|