Characterization of the conformational dynamics of the Nek2 leucine zipper domain
thesisposted on 12.03.2013, 15:21 by Rebecca Alice Croasdale
Nek2 is a cell cycle regulated protein kinase. Its expression and activity peak in S and G2 phase before the protein is targeted for degradation in mitosis. Nek2 activity promotes centrosome separation and bipolar spindle formation, processes that are essential for maintaining the fidelity of chromosome segregation and cell division. Importantly, Nek2 inhibition results in increased apoptosis and senescence in cancer cell lines and tumour xenografts making Nek2 an attractive target for chemotherapeutic intervention. The α-helix is the most common secondary structure in proteins, with 2-3% of all proteins adopting a coiled-coil structure. The coiled-coil motif mediates a diverse range of functions including DNA transcription, intracellular protein shuttling, membrane signalling, and coordination of the cell cycle. Nek2 contains two coiled-coil motifs in its C-terminal non-catalytic domain, the first of which forms a leucine zipper structure, whilst the second has recently been classified as a SARAH domain. The leucine zipper spans residues 304-340 and is responsible for Nek2 dimerization, autophosphorylation and activation. The Nek2 leucine zipper contains an unusual pattern of charged residues in the dimerization interface. Through studies on isolated fragments, we found that the Nek2 leucine zipper exists primarily as a dimer in solution, albeit with concentration dependent higher order oligomerization. However, the positioning of charged residues enabled the helices to undergo a register shift between two alternative heptad conformations on a timescale of 17s-1. This represents slow-intermediate exchange on the NMR timescale, greatly increasing the transverse and longitudinal relaxation rates leading to enhanced loss of signal intensity. As this precluded structure determination through NMR studies on wild-type fragments, a K309C mutant was generated that ‘locked’ the leucine zipper into one conformation. The significantly reduced relaxation rates confirming the presence of conformational dynamics and making structural determination possible in the future.