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 β-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-β-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 β-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. 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 β-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-β-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 β-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 Aβ peptide (1Selkoe D.J. Schenk D. Annu. Rev. Pharmacol. Toxicol. 2003; 43: 545-584Crossref PubMed Scopus (754) Google Scholar) or of the microtubule (MT) 1The abbreviations used are: MT, microtubule; PHF, paired helical filament; MES, 4-morpholineethanesulfonic acid; Pipes, 1,4-piperazinediethanesulfonic acid; HSQC, heteronuclear single quantum correlation. -associated tau protein (2Mandelkow E.M. Mandelkow E. Trends Cell Biol. 1998; 8: 425-427Abstract Full Text Full Text PDF PubMed Scopus (445) Google Scholar). 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 Aβ 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 (3Gustke N. Trinczek B. Biernat J. Mandelkow E.M. Mandelkow E. Biochemistry. 1994; 33: 9511-9522Crossref PubMed Scopus (547) Google Scholar). The affinity is regulated by phosphorylation particularly at KXGS-motifs in the repeats (4Biernat J. Gustke N. Drewes G. Mandelkow E.M. Mandelkow E. Neuron. 1993; 11: 153-163Abstract Full Text PDF PubMed Scopus (662) Google Scholar). Interestingly the same phosphorylation sites have an inhibitory influence on aggregation (5Schneider A. Biernat J. von Bergen M. Mandelkow E. Mandelkow E.M. Biochemistry. 1999; 38: 3549-3558Crossref PubMed Scopus (460) Google Scholar). 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 (6Barghorn S. Mandelkow E. Biochemistry. 2002; 41: 14885-14896Crossref PubMed Scopus (286) Google Scholar)). 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 Fourier-transform infrared spect