Proline-directed Pseudo-phosphorylation at AT8 and PHF1 Epitopes Induces a Compaction of the Paperclip Folding of Tau and Generates a Pathological (MC-1) Conformation

Abstract

Tau, a neuronal microtubule-associated protein that aggregates in Alzheimer disease is a natively unfolded protein. In solution, Tau adopts a "paperclip" conformation, whereby the N-and C-terminal domains approach each other and the repeat domain (Jeganathan, S., von Bergen, M., Brutlach, H., Steinhoff, H. J., and Mandelkow, E. (2006) Biochemistry 45, 2283-2293). In AD, Tau is in a hyperphosphorylated state. The consequences for microtubule binding or aggregation are a matter of debate. We therefore tested whether phosphorylation alters the conformation of Tau. To avoid the ambiguities of heterogeneous phosphorylation we cloned "pseudo-phosphorylation" mutants of Tau where combinations of Ser or Thr residues were converted into Glu. These mutations were combined with FRET pairs inserted in different locations to allow distance measurements. The results show that the paperclip conformation becomes tighter or looser, depending on the pseudo-phosphorylation state. In particular, pseudo-phosphorylation at the epitope of the diagnostic antibody AT8* (S199E ؉ S202E ؉ T205E) moves the N-terminal domain away from the C-terminal domain. Pseudophosphorylation at the PHF1 epitope (S396E ؉ S404E) moves the C-terminal domain away from the repeat domain. In both cases the paperclip conformation is opened up. By contrast, the combination of AT8* and PHF1 sites leads to compaction of the paperclip, such that the N-terminus approaches the repeat domain. The compaction becomes even stronger by combining pseudo-phosphorylated AT8*, AT100, and PHF1 epitopes. This is accompanied by a strong increase in the reaction with conformation-dependent antibody MC1, suggesting the generation of a pathological conformation characteristic for Tau in AD. Furthermore, the compact paperclip conformation enhances the aggregation to paired helical filaments but has little influence on microtubule interactions. The data provide a framework for the global folding of Tau dependent on proline-directed phosphorylation in the domains flanking the repeats and the consequences for pathological properties of Tau.Microtubules that serve as the tracks for motor proteins are important for the intracellular transport of vesicles, organelles, and protein complexes by motor proteins (2, 3). Microtubule dynamics are modulated by microtubule-associated proteins that bind to the surface of microtubules; among these, Tau protein is one of the major microtubule-associated proteins in neurons (4, 5). Its expression is strongly up-regulated during neuronal development to promote the generation of cell processes and to establish cell polarity (6). During this phase, Tau becomes sorted into the axon, and it diversifies into 6 different isoforms by alternative splicing (7,8). In Alzheimer disease, Tau becomes hyperphosphorylated, missorted into the somatodendritic compartment, and aggregates into neurofibrillary tangles (9).The numerous phosphorylation sites of Tau (10) can be broadly subdivided into three classes: (i) SP/TP motifs in the flanking regions of the repeat doma...

Aggregation of Detergent-insoluble Tau Is Involved in Neuronal Loss but Not in Synaptic Loss

“…When AT8 sites are pseudo-phosphorylated, the distance between the tau N and C termini is longer than when PHF1 sites or AT8 and PHF1 sites are pseudo-phosphorylated (28). For the latter situations, the distances between the tau C and N termini are similar.…”

“…Pseudophosphorylation of either the AT8 site or the PHF1 site causes the paperclip conformation to open up, whereas pseudophosphorylation of both AT8 and PHF1 sites causes tau to form a compact paperclip conformation (28). Additional phosphorylation induces pathological conformational changes (28).…”

Structural Impact of Proline-Directed Pseudophosphorylation at AT8, AT100, and PHF1 Epitopes on 441-Residue Tau

“…A weakened contact between the two termini is in agreement with a lower fluorescence resonance energy transfer (FRET) efficiency between residues 432 and 17 in the E-mutant relative to wt tau. 9 However, the FRET efficiencies between residues 310 and 17 and between residues 432 and 322 were larger in the E-mutant than in the wt protein, 9 in apparent contrast to the NMR PRE profiles. We attribute the differences to the use of two hydrophobic labels (tryptophan and dansyl group) in the FRET studies.…”

Tau Monoclonal Antibody Generation Based on Humanized Yeast Models

“…Phosphorylation of Thr 231 by Gsk-3␤ is known to prevent Tau from binding microtubules (4) and to relieve the inhibitory activity of the N terminus over the C terminus of Tau so as to allow kinases such as Gsk-3␤ to access and subsequently phosphorylate Tau at other epitopes (48). Indeed, monomeric Tau is thought to adopt a so-called "paperclip" conformation when in solution, where the C-terminal end of Tau folds over the MTBR domain and the N terminus folds back to come in close proximity to the C terminus (49,50). It is conceivable that the opening of this paperclip conformation is a first essential step for Tau oligomerization.…”