Sites of Tau Important for Aggregation Populate β-Structure and Bind to Microtubules and Polyanions

Abstract

The aggregation of the microtubule-associated tau protein and formation of "neurofibrillary tangles" is one of the hallmarks of Alzheimer disease. The mechanisms underlying the structural transition of innocuous, natively unfolded tau to neurotoxic forms and the detailed mechanisms of binding to microtubules are largely unknown. Here we report the high-resolution characterization of the repeat domain of soluble tau using multidimensional NMR spectroscopy. NMR secondary chemical shifts detect residual ␤-structure for 8 -10 residues at the beginning of repeats R2-R4. These regions correspond to sequence motifs known to form the core of the cross-␤-structure of tau-paired helical filaments. Chemical shift perturbation studies show that polyanions, which promote paired helical filament aggregation, as well as microtubules interact with tau through positive charges near the ends of the repeats and through the ␤-forming motifs at the beginning of repeats 2 and 3. The high degree of similarity between the binding of polyanions and microtubules supports the hypothesis that stable microtubules prevent paired helical filament formation by blocking the tau-polyanion interaction sites, which are crucial for paired helical filament formation.Alzheimer disease is characterized by abnormal protein deposits in the brain, such as amyloid plaques or neurofibrillary tangles, formed by fibrous assemblies of the A␤ peptide (1) or of the microtubule (MT) 1 -associated tau protein (2). These aggregates are thought to be toxic to neurons, either by causing some toxic signaling defect or by obstructing the cell interior. Therefore, one of the top priorities in Alzheimer research is to understand the reasons for the pathological aggregation and to find methods to prevent it. Although the structural principles governing A␤ aggregation are known in some detail, little is known for the tau protein.Tau is a microtubule-associated protein that regulates MT stability, neurite outgrowth, and other MT-dependent functions. The three or four repeats in the C-terminal half of the protein and the flanking proline-rich basic domains are known to be involved in MT binding (3). The affinity is regulated by phosphorylation particularly at KXGS-motifs in the repeats (4). Interestingly the same phosphorylation sites have an inhibitory influence on aggregation (5). Unbound tau can assemble into Alzheimer-like paired helical filaments (PHFs) whose polymerization can be enhanced by oxidation of SH groups and by polyanions (e.g. heparin, poly-Glu (6)). On the other hand, tau has a hydrophilic character, is highly soluble, and belongs to the class of natively unfolded proteins with no apparent ordered secondary structure detectable by far-UV CD or Fouriertransform infrared spectroscopy (7,8). Therefore, it is unclear why tau should aggregate in a specific manner and what structural principles could be responsible for this.Tau can aggregate as an intact protein, 352-441 residues in length (depending on isoform), so that all six tau isoforms are found in Alzheim...

Biochemistry and Cell Biology of Tau Protein in Neurofibrillary Degeneration

“…The amino-terminal fragment does not bind to microtubules but projects away from the microtubule surface and hence is termed "projection domain" (Hirokawa et al 1988). A more detailed analysis, based on nuclear magnetic resonance (NMR) spectroscopy, confirms and refines these features: Pronounced interactions with microtubules (MT) occur beween Tau residues 200 and 400 (Mukrasch et al 2005;Sillen et al 2007) and thus cover the repeat domain plus the adjacent flanking domains of 40 residues each, consistent with binding studies (see below). However, weaker interactions are also distributed elsewhere in the carboxy-terminal half of Tau (Mukrasch et al 2009).…”

“…Tau binds strongly to the MT surface, but surprisingly does not self-assemble; rather, the MT surface can be overloaded with a coat of Tau molecules in a nonfilamentous form . Since the Tau-Tau interaction site largely overlaps with the Tau-MT interaction site (Mukrasch et al 2005), it appears that MTs override Tau's capacity for fibrillization, perhaps by stabilizing a nonaggregant conformation. Thus, MTs act effectively as chaperones for Tau to prevent their abnormal aggregation.…”

Quantitative mapping of microtubule-associated protein 2c (MAP2c) phosphorylation and regulatory protein 14-3-3ζ-binding sites reveals key differences between MAP2c and its homolog Tau

“…Positively charged regions of Tau are known to bind to acidic residues of tubulin and polyanions (49,50), and MAP2c is likely to behave in a similar manner. We propose that the stretches of positively charged residues in MAP2c and Tau are also involved in the interaction with electronegative regions within the highly acidic 14-3-3 protein (pI Ϸ 4.7).…”

Clinical and pathological features of an Alzheimer's disease patient with the MAPT ΔK280 mutation

“…In many ways, the in vitro behaviour of the mutation was anomalous; it was the only mutation which increased the proportion of 3R-tau and while cDNA transfection showed abnormalities in tubulin binding, this work has uncertain relevance to the human situation because the mutation is in the potentially excluded exon 10. Despite this uncertainty, the ΔK280 protein variant has been extensively used in biochemical experiments designed to understand the effects of the "FTDP-17 mutations" on tau biophysics, tau aggregation and on tubulin binding (Barghorn et al, 2000, Mukrasch et al, 2005Vogelsberg-Ragaglia et al,. 2005;von Bergen et al, 2001).…”

“…The other authors declare no conflicts of interest. tau transcript, which would be predicted to occur in very small amounts since the mutation is in exon 10 and thus would be expected to be largely self-excluding, is extremely fibrillogenic and has frequently been used to model tau aggregation (Barghorn et al, 2000;Mukrasch et al, 2005;Vogelsberg-Ragaglia et al, 2005;von Bergen et al, 2001). Furthermore, it has been demonstrated to strongly reduce the ability of tau to promote microtubule assembly (Rizzu et al, 1999).…”