Evaluation of protein kinases for solution NMR spectroscopy and the structural mechanism of inhibition and activation of an oncogenic calcium calmodulin dependent protein kinase

Tong, Michael (2012). Evaluation of protein kinases for solution NMR spectroscopy and the structural mechanism of inhibition and activation of an oncogenic calcium calmodulin dependent protein kinase. University of Birmingham. Ph.D.

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Abstract

Protein kinases are important mediators of cell signalling that are often implicated in disease when deregulation occurs. The catalytic kinase domain is highly conserved with 518 identified members in the super family. Kinase structural studies are mainly based on relatively static crystal structures. However protein kinases are inherently dynamic entities in solution. Several Ser/Thr protein kinases were evaluated by NMR in order to obtain an amenable target for solution structure and functional characterisation. Subsequently a calcium calmodulin dependent protein kinase dubbed CaMK1D was identified as the optimal system. CaMK1D normally mediates intracellular signalling downstream of chemokines. It is amplified in breast cancer, and induces cell proliferation, migration and invasion. Here we report the backbone resonance assignments for the 38 kDa human autoinhibited CaMK1D in its free state, encompassing a canonical bi-lobed kinase fold and autoinhibitory and calmodulin binding domains. These assignments allowed us to probe the binding mode of CaMK1D with small molecule ligands and refine the crystal structure via dihedral angle restraints for a more complete structure. Furthermore we investigated the solution structure of the CaMK1D∙Ca\(^{2+}\)/CaM complex and propose a model of the activation mechanism and establish a key residue implicated in complex formation.

Type of Work:

Thesis (Doctorates > Ph.D.)

Award Type:

Doctorates > Ph.D.

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