Structural characterization on the complex of calmodulin and the regulatory domain of calcineurin

Autor: Sin-Mao Lin, 林欣楙
Rok vydání: 2017
Druh dokumentu: 學位論文 ; thesis
Popis: 105
Calcineurin (CaN) is a Ca2+ /CaM-dependent Ser/Thr protein phosphatase. CaN plays important roles in neuronal development and function, immune activation and cardiac growth. CaN is a heterodimer composed of a 60 kDa A-subunit and 19 kDa B- subunit. A subunit consists with catalytic domain, B subunit binding helix, regulatory domain (RD) and auto-inhibitory domain (AID). The sequence of B subunit has 35% homologous to the CaM, and structurally homologous to CaM. Therefore, B subunit can bind to four calcium ions. In absence of Ca2+, catalytic domain is blocked by auto-inhibitory domain and the enzyme is inactive. Upon binding to CaM, CaN undergoes a conformational rearrangement, the auto inhibitory domain is displaced and thus allows for full activity. We have successfully cloned three truncated forms of CaN A subunit (CaN391-414, CaN369-468 and CaN369-491). To elucidate the regulatory role of CaM on CaN, and to gain more insight into the interactions between CaM and CaN from the structural point of view, we have applied nuclear magnetic resonance (NMR) and circular dichroism (CD) techniques to study these structures of CaM bound with these truncated forms. Circular dichroism spectrum demonstrated that these three truncated forms of CaN A subunit exhibited mostly random coil structures in their free forms. However, the secondary structure of these three truncated forms of CaN A subunit, especially α-helical structure increased upon association with CaM. The full resonance assignments and structure of CaM with CaN391-414 have been obtained. The structure of free CaN369-491 was characterized by NMR spectroscopy and the backbone assignment of 15N-CaN369-491/CaM complex was completed. We propose the model of CaN369-491/CaM complex.
Databáze: Networked Digital Library of Theses & Dissertations
Externí odkaz: