Abstract
Phosphorylation of the neuronal Tau protein is implicated in both the regulation of its physiological function of microtubule stabilization and its pathological propensity to aggregate into the fibers that characterize Alzheimer's diseased neurons. However, how specific phosphorylation events influence both aspects of Tau biology remains largely unknown. In this study, we address the structural impact of phosphorylation of the Tau protein by Nuclear Magnetic Resonance (NMR) spectroscopy on a functional fragment of Tau (Tau[Ser208โSer324] = TauF4). TauF4 was phosphorylated by the prolineโdirected CDK2/CycA3 kinase on Thr231 (generating the AT180 epitope), Ser235, and equally on Thr212 and Thr217 in the Prolineโrich region (Tau[Ser208โGln244] or PRR). These modifications strongly decrease the capacity of TauF4 to polymerize tubulin into microtubules. While all the NMR parameters are consistent with a globally disordered Tau protein fragment, local clusters of structuration can be defined. The most salient result of our NMR analysis is that phosphorylation in the PRR stabilizes a short ฮฑโhelix that runs from pSer235 till the very beginning of the microtubuleโbinding region (Tau[Thr245โSer324] or MTBR of TauF4). Phosphorylation of Thr231/Ser235 creates a Nโcap with helix stabilizing role while phosphorylation of Thr212/Thr217 does not induce modification of the local transient secondary structure, showing that the stabilizing effect is sequence specific. Using paramagnetic relaxation experiments, we additionally show a transient interaction between the PRR and the MTBR, observed in both TauF4 and phosphoโTauF4. Proteins 2012. ยฉ 2011 Wiley Periodicals, Inc.