Sites of Tau Important for Aggregation Populate β-Structure and Bind to Microtubules and Polyanions
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 spectroscopy (7Schweers O. Schonbrunn-Hanebeck E. Marx A. Mandelkow E. J. Biol. Chem. 1994; 269: 24290-24297Abstract Full Text PDF PubMed Google Scholar, 8von Bergen M. Friedhoff P. Biernat J. Heberle J. Mandelkow E.M. Mandelkow E. Mol. Biol. Cell. 2000; 11: 363AGoogle Scholar). 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 Alzheimer PHFs (9Buee L. Hamdane M. Delobel P. Sambo A.V. Begard S. Ghestem A. Sergeant N. Delacourte A. J. Soc. Biol. 2002; 196: 103-108Crossref PubMed Scopus (7) Google Scholar). The isoforms differ by two inserts near the N-terminal end and the presence of either four or three imperfect repeat sequences in the C-terminal half of the protein (see Fig. 1). The region comprising the repeat sequences forms the core of PHFs (10Wischik C.M. Novak M. Thogersen H.C. Edwards P.C. Runswick M.J. Jakes R. Walker J.E. Milstein C. Roth M. Klug A. Proc. Natl. Acad. Sci. U. S. A. 1988; 85: 4506-4510Crossref PubMed Scopus (821) Google Scholar) and also promotes PHF assembly in vitro (11Wille H. Drewes G. Biernat J. Mandelkow E.M. Mandelkow E. J. Cell Biol. 1992; 118: 573-584Crossref PubMed Scopus (436) Google Scholar, 12Friedhoff P. von Bergen M. Mandelkow E.M. Davies P. Mandelkow E. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 15712-15717Crossref PubMed Scopus (295) Google Scholar). For PHF aggregation two hexapeptides at the beginning of the second and third repeats (275VQI-INK280 and 306VQIVYK311) are crucial. These are able to initiate the aggregation of tau into bona fide paired helical filaments with cross-β-structure and thereby represent minimal tau-tau interaction motifs (13von Bergen M. Barghorn S. Li L. Marx A. Biernat J. Mandelkow E.M. Mandelkow E. J. Biol. Chem. 2001; 276: 48165-48174Abstract Full Text Full Text PDF PubMed Scopus (454) Google Scholar, 14Goux W.J. Kopplin L. Nguyen A.D. Leak K. Rutkofsky M. Shanmuganandam V.D. Sharma D. Inouye H. Kirschner D.A. J. Biol. Chem. 2004; 279: 26868-26875Abstract Full Text Full Text PDF PubMed Scopus (190) Google Scholar). Because tau is a highly flexible protein, it has resisted all attempts at crystallization for a high-resolution x-ray structure. X-ray analyses exist only in the form of solution scattering (confirming the unfolded nature of the protein, (7Schweers O. Schonbrunn-Hanebeck E. Marx A. Mandelkow E. J. Biol. Chem. 1994; 269: 24290-24297Abstract Full Text PDF PubMed Google Scholar)) and fiber diffraction (confirming the cross-β-structure of PHFs, (13von Bergen M. Barghorn S. Li L. Marx A. Biernat J. Mandelkow E.M. Mandelkow E. J. Biol. Chem. 2001; 276: 48165-48174Abstract Full Text Full Text PDF PubMed Scopus (454) Google Scholar)). We have now applied NMR spectroscopy as an alternative to structural Here we report the high-resolution characterization of the repeat domain of in which either all four repeats are or repeat two has to the and tau isoforms and (see Fig. 1). show that the repeat domain of soluble tau regions of residual β-structure that have the to as for aggregation of tau into PHFs, and we residues involved in the interaction with and with polyanions that promote of Tau and tau in the of in as (3Gustke N. Trinczek B. Biernat J. Mandelkow E.M. Mandelkow E. Biochemistry. 1994; 33: 9511-9522Crossref PubMed Scopus (547) Google Scholar)) (see Fig. 1). all four repeats of the tau is the second to tau The proteins by of the of the protein and by the tau proteins with and stable the E. protein in a minimal with and and the E. protein on on with and in by E. in minimal of The cell in MES, with a The with a cell and for The soluble by the two of MES, 2 and on an The proteins by a of MES, 2 NMR or protein in with of as E.M. M. U. J. Mol. Biol. PubMed Scopus Google Scholar) and at in MT assembly Pipes, in the presence of at for of the polymerization for at to a of on for with two of and with for The in a at NMR at on and NMR NMR and using S. G. J. A. J. PubMed Scopus Google Scholar) and 3. to for the of and shift as the between the chemical shifts and the for the at S. J. J. Chem. Soc. 2001; PubMed Scopus Google Scholar). for and 1994; PubMed Scopus Google as the chemical shifts of residues are particularly to and the in the studies by 1994; PubMed Scopus Google Scholar) is to the one used the in of the secondary shifts by the secondary shift for that in a 1994; PubMed Scopus Google and by the of residues in the with protein and and and at and poly-Glu in formation by for and with and with and with and and with For NMR and and formation at and for MT of and in the and and in the chemical shift for and chemical shifts binding using and are the between the and chemical of and in of the tau repeat domain and with at and The and only a of chemical a high degree of and unfolded in with CD and (7Schweers O. Schonbrunn-Hanebeck E. Marx A. Mandelkow E. J. Biol. Chem. 1994; 269: 24290-24297Abstract Full Text PDF PubMed Google Scholar, Bergen M. Barghorn S. Li L. Marx A. Biernat J. Mandelkow E.M. Mandelkow E. J. Biol. 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These shifts show for and the of six of secondary chemical shifts found for residues and to the of β-structure These at the beginning of repeats and motifs and the motifs and known to be for the abnormal aggregation of tau into PHFs Bergen M. Friedhoff P. Biernat J. Heberle J. Mandelkow E.M. Mandelkow E. Mol. Biol. Cell. 2000; 11: 363AGoogle Scholar). of secondary chemical shifts for a of and Fig. is that the of secondary chemical shifts at the beginning of the repeats and the of is of a to form β-structure of the of three the secondary chemical shifts in region with the that of residues show chemical shifts to the of an in the the of the be with of the presence of only three residues of the C-terminal is for repeat is that the repeats represent structural of the and secondary chemical shifts that are and of the for residues and the regions in the beginning of residues and in repeats and show a of secondary a β-structure that no β-structure is in the region of repeat of the presence of a The regions of high for the β-structure are by the three show positive and secondary chemical shifts in all four either a helical or a in with the that and sequences in or are the motifs in the is also in The ends of the regions of high β-structure are by positive secondary chemical shifts of the in repeat into that sequence such as and motifs high regions in and are in the and secondary chemical shifts a at the C-terminal end of the regions of high β-structure 8–10 residues at the beginning of repeats show a residual β-structure between regions with a high The β-structure is at the of the third in the second and repeat it is a of These in and are in with The secondary chemical for is to that in that the of the second repeat the β-structure in the of the repeat domain of of and and of of of the interaction and binding D.J. Mol. Biol. Google Scholar). The binding of the polyanions and poly-Glu to and by The of the chemical shift for on the binding to the high chemical shift of some residues by a of the of chemical shift on the NMR the of the residues the in the of at the beginning of the second repeat chemical shift in the of and and The of and residues that the binding to polyanions is of with a of chemical shift that the binding for the two of polyanions is repeat 2 is and can to the interaction with the same and residues as in chemical shift for the six residues in the third repeat that are for tau aggregation to PHFs and with the chemical shift and the of some at high by the in the with for The is to aggregation, that at the end of the of as by and in the the of aggregation we a of in which the of between and and the for at to and of and with a of at the to and at for 2 the 2 the to and an such that the protein at for in The of the chemical shift that in Fig. The for residues by of with the of residues chemical shift the 2 at the of all to with the no in the protein that particularly an of the same the chemical shift that by of largely the 2 at with a of highly to the one of of in Fig. and at the in to and with the heparin, chemical shift to the 2 at the with of or the protein to at the end of the that of These that the chemical shift of the binding of the to the protein and of the protein to high of for or to high at a of of the protein the chemical shift which by of heparin, largely aggregation, to the thereby the of in The of and to Tau binding of and to characterized using the NMR chemical shift perturbation D.J. Mol. Biol. Google Scholar) in which of and in the presence of of The influence of by the NMR at and the of the at the of tau with characterized by and assembly at the the of the NMR the is for one at to the of in the presence of and the presence of tau the of a in (see G. P. H. Mandelkow E.M. Mandelkow E. A. J. Mol. Biol. 2004; PubMed Scopus Google Scholar)) For a the and the and by and MT assembly with and at or is found in the of to the binding in of in the The same for These show that the the of the NMR and that binding of and are in with (3Gustke N. Trinczek B. Biernat J. Mandelkow E.M. Mandelkow E. Biochemistry. 1994; 33: 9511-9522Crossref PubMed Scopus (547) Google Scholar). chemical shift of residues by the of the at the at high MT of an of or to to the NMR of or are of the in found in the of and and that repeat a binding for to with the nature of the repeat are at in N-terminal to the The same and at to a the chemical shift for residues at the beginning of the second repeat region and with and the chemical shift only by the of the only chemical shift for in the beginning of repeat 3. of to on the other hand, a on residues chemical shift for residues in the and Fig. for the of the N-terminal of the third repeat into binding to the of the for MT The for residues as as that of the of the second the that the chemical shift of to and the for other residues in the Fig. the of chemical shift by of for and and We that repeats of tau are to the positive charges of the The of repeat one is by the positive of the region is for binding to is in the beginning of the third which it is particularly for binding of tau to Tau is an protein tau is one of the microtubule-associated proteins in the it a in the formation of and is for cell and tau is a of the class of or proteins whose are by x-ray a tau one of the hallmarks of Alzheimer the form of it forms pathological protein aggregates that are toxic to the These reasons to of the multidimensional NMR with the of the structural of tau that are responsible for and pathological the we on the repeat domain of tau that forms the core of the domain as well as the core of Alzheimer paired helical filaments We have used NMR spectroscopy to detect structural motifs and of aggregation in the repeat region of tau and to interaction with and polyanions that are known to promote the aggregation into The can be as The beginning of repeat is characterized by a of 8–10 residues with secondary chemical shift For the of a high of These regions with sequence motifs known to be involved in the abnormal aggregation of The β-forming motifs are between motifs that have a high for and the (e.g. the of of the motifs in repeats and The are the same in of tau with or repeats to or that the repeats and other binding of tau show regions of the beginning of repeats and with the and of high positive of the supports the that the abnormal aggregation by is on the same structural as the interaction with Tau of a high-resolution characterization by it to the of all the NMR of single at only of the repeat domain of These for residual structure C. A. A. G. 2004; PubMed Scopus Google Scholar). the high degree of by the of a well secondary and we able to the of and NMR chemical shifts are for the of of the that of and at the beginning of in with the of and at the beginning of in the chemical shifts are by the sequence or residual structure is in and the beginning of repeats and a for β-structure of 8–10 residues could be and These on ends by motifs with a high for such as the motifs or the or The β-structure is for residues in the beginning of that is an for all residues and it that all residues β-structure at the same secondary chemical shifts for residues and a for β-structure in the C-terminal half of repeats and These are in with other secondary structure that tau has a of secondary structure (7Schweers O. Schonbrunn-Hanebeck E. Marx A. Mandelkow E. J. Biol. Chem. 1994; 269: 24290-24297Abstract Full Text PDF PubMed Google Scholar)). in an NMR of D. P. M. D. Li L. Biochemistry. 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M. 1998; PubMed Scopus Google Scholar). Therefore, of the detailed by which tau to is highly the we have characterized the binding of tau to on the of tau Because it has that the repeat domain of tau is for MT binding we high-resolution to region of the protein. The NMR residues as an region of tau for binding to is in with analyses by M. J. Biol. Chem. 2000; Full Text Full Text PDF PubMed Scopus Google Scholar, J. Cell Biol. 1994; PubMed Scopus Google Scholar). and of MT binding which or to well with the chemical shift for and of only chemical shift with a to binding affinity as by the N-terminal of the NMR show that the interaction is enhanced by interaction of positive charges of the in MT binding we also the interaction of with it that the C-terminal sequences of the repeat region a to MT binding affinity in in tau have no The C-terminal the NMR on a and can be used to for in the MT The highly of chemical shift in and of that the binding sites of the to the the nature of the tau binding sites on and is that tau with the of that is to be on the of E. Cell 1999; PubMed Scopus Google Scholar). of chemical shift for and of with of polyanions a binding that poly-Glu the C-terminal sequence of the NMR that the to the of The binding of tau to and polyanions also supports the hypothesis that stable prevent PHF formation by blocking the tau-polyanion interaction sites, which are crucial for PHF formation. is known that phosphorylation at motifs affinity of tau to (4Biernat J. Gustke N. Drewes G. Mandelkow E.M. Mandelkow E. Neuron. 1993; 11: 153-163Abstract Full Text PDF PubMed Scopus (662) Google Scholar). We have that the repeat domain of tau to through of high positive comprising the beginning of and as well as regions of the and are by only four residues regions two to three positive charges at the C-terminal end of the of phosphorylation can be the of the of the the positive for MT be by the high of the protein of which the of positive and charges to the high of tau be to a detailed of the interaction by the between the and the region a the for which for residues and and the between the interaction sites in a that no to interaction sites on the MT The a detailed of the of the repeat region of the microtubule-associated protein tau and interaction with polyanions and The presence of of aggregation at the beginning of repeats and which with the for β-structure and the binding regions of tau to and polyanions, the of regions for MT and for in the in which tau is into Alzheimer paired helical filaments. The in be or to the aggregation of tau in neurons, and to prevent one of the crucial in disease. We and for for with the of chemical shift and Mandelkow for the The the of the for of the in with