Description
- Abstract:
- Ser/Thr phosphatases dephosphorylate their targets with high specificity, yet the structural determinants of substrate recognition and dephosphorylation are poorly understood. Calcineurin (CN) is a conserved Ca2+/calmodulin-dependent Ser/Thr phosphatase that regulates many cellular functions and is the direct target of the immunosuppressant drugs FK506 and cyclosporin A (CSA). CN dephosphorylates the NFAT family of transcription factors, which then activate transcription of genes required for various cellular functions. This dissertation describes the structural basis for CN-substrate recognition and provides insight into the molecular mechanisms used by CN inhibitors. <br/> <br/> To investigate CN-substrate recognition we used X-ray crystallography, biochemistry, molecular dynamics simulations and in vivo experiments to study A238L, a viral protein inhibitor of CN and a substrate mimic. The 1.7 Å structure of the A238L-CN complex provides the first structural description of how CN recognizes substrates via the conserved "LxVP" motif, and enabled molecular dynamics simulations that provided the first description of substrate dephosphorylation by a Ser/Thr phosphatase. The structure also revealed that A238L competitively inhibits CN by occupying the PxIxIT and LxVP substrate recognition sites to prevent substrate docking, leaving the catalytic center fully accessible. This study establishes that FK506 and CSA also bind to the CN LxVP site to prevent LxVP-mediated substrate recognition. This highlights the importance of this interaction for substrate dephosphorylation and provides a structural basis for the development of new CN inhibitors. <br/> <br/> To gain further insight into CN regulation, we used NMR spectroscopy and isothermal titration calorimetry to characterize CN inhibition by the endogenous regulator RCAN1. NMR studies of free RCAN1 showed that it belongs to a family of proteins known as intrinsically disordered proteins, which are characterized by their lack of tertiary structure. However, we identified two partially populated α-helices in RCAN1, which are likely important for the ability of RCAN1 to act as both an inhibitor and an enhancer of CN activity. RCAN1 is overexpressed in individuals with Down syndrome, and its inhibition of CN directly correlated with symptoms of this disease. Therefore, these findings significantly advance our understanding of the underlying mechanism of Down syndrome.
- Notes:
- Thesis (Ph.D. -- Brown University (2013)
Access Conditions
- Rights
- In Copyright
- Restrictions on Use
- Collection is open for research.
Citation
Grigoriu, Simina r.,
"The Molecular Basis of Calcineurin Inhibition by Protein Regulators"
(2013).
Molecular Biology, Cell Biology, and Biochemistry Theses and Dissertations.
Brown Digital Repository. Brown University Library.
https://doi.org/10.7301/Z0765CPQ
Relations
Collection:
-
Molecular Biology, Cell Biology, and Biochemistry Theses and Dissertations
Theses and Dissertations for the Molecular Biology, Cell Biology, and Biochemistry department....