Bibow, S.

Biernat, J.

Dürr, U.

Mandelkow, E.

Narayanan, R.

Zweckstetter, M.

English

MPG.PuRe

Automatic Assignment of the Intrinsically Disordered Protein Tau with441-ResiduesRhagavendran L. Narayanan,† Ulrich H. N. Dürr,† Stefan Bibow,† Jacek Biernat,§Eckhard Mandelkow,§ and Markus Zweckstetter*,†,‡Department of NMR-based Structural Biology, Max Planck Institute for Biophysical Chemistry, Am Fassberg 11,37077 Goettingen, Germany, DFG Center for the Molecular Physiology of the Brain, 37073 Goettingen, Germany,and Max Planck Unit for Structural Molecular Biology, c/oDESY, Notkestrasse 85, 22607 Hamburg, GermanyReceived June 28, 2010; E-mail: mzwecks@gwdg.deAbstract: Intrinsically disordered proteins carry out many impor-tant functions in the cell. However, the lack of an ordered structurecauses dramatic signal overlap and complicates the NMR-basedcharacterization of their structure and dynamics. Here we dem-onstrate that the resonance assignment of 441-residue Tau andits smaller isoforms, htau24 (383 residues) and htau23 (352residues), three prototypes of intrinsically disordered proteins,which bind to microtubules and play a key role in Alzheimerdisease, can be obtained within 5 days by a combination of seven-dimensional NMR spectra with optimized methods for automaticassignment. Chemical shift differences between the three iso-forms provide evidence for the global folding of Tau in solution.In the past decade intrinsically disordered proteins (IDPs)obtained significant attention as more than 30% of eukaryoticproteins comprise unstructured regions larger than 50 consecutiveresidues.1 A hallmark of IDPs is the lack of a well-structured three-dimensional fold.2 Despite the absence of a rigid secondary ortertiary structure, IDPs carry out many important functions in thecell, for example the regulation of transcription and translation, thestorage of small molecules, and the regulation of the self-assemblyof large multiprotein complexes.3 Tau, a prototype of IDPs, bindsto microtubules and plays a key role in Alzheimer disease.4Since disordered proteins tend to be highly flexible and havevariable conformations, they have mostly not been amenable forstructure analysis by crystallography. Thus NMR spectroscopy isthe only method that allows a description of their conformationsand dynamics with high resolution.5 The lack of an orderedstructure, however, causes dramatic signal overlap limiting previousNMR investigations of IDPs to around 200 residues (e.g., refs 6,7). For the largest Tau isoform with 441 residues, htau40 (Figure1), the number of overlapping signals is 3.5-fold higher than thatin the largest currently assigned globular protein.8 In addition, thelarge number of proline residues, the strongly repetitive primarysequence in the repeat domains, and the complexity of the sequencefurther complicate the resonance assignment.8,9 Only as part of anongoing project taking several months to years and building onthe assignment of smaller fragments of Tau in a “divide-and-conquer” strategy, we were recently able to assign the backboneresonances of the full-length protein.8Here we demonstrate that the resonance assignment of 441-residue Tau and its smaller isoforms, htau24 (383 residues) andhtau23 (352 residues) (Figure 1), can be obtained within 5 days bya combination of seven-dimensional NMR spectra with optimizedmethods for automatic assignment. Chemical shift differencesbetween the three isoforms provide evidence for the global foldingof Tau in solution.Due to the lack of stable hydrogen bonds, NMR spectra of IDPsare sensitive to solvent exchange. At pH 6.8 an increase in temperaturefrom 5 to 35 °C results in the broadening of about 20% of backboneresonances of Tau beyond detection in 2D 1H,15N HSQC spectra(Supporting Information, Supporting Figure 1). This motivated the useof HR-detection and direct carbon detection for the assignment ofIDPs.7,10,11 However, when we reduced the pH to 6.0 the 1H/15Nsignals of Tau were not attenuated at higher temperatures but ratherincreased by about 40% due to reduced amide proton exchange, morerapid tumbling, and improved relaxation properties. We thus asked ifthe favorable relaxation properties help to acquire very high-dimensional NMR spectra.Previously, we obtained the backbone resonance assignment ofTau using 3D HACANNH and 3D HNN experiments at 5 °C, pH6.8.8,12,13 Here we recorded 5D HACACONH and 7D HNCO-(CA)CBCANH experiments at 25 °C, pH 6.0. To avoid prohibi-tively long measurement times, we employed simultaneous evo-lution of multiple indirect dimensions14 as proposed in automaticprojection reconstruction spectroscopy (APSY).15 The 7D APSYHNCO(CA)CBCANH correlates in a single cross peak sevendifferent chemical shifts (HN(i), N(i), CO(i-1), C(i-1), CR(i-1),N(i-1), HN(i-1)) and sequentially connects residues through Nand HN frequencies (Supporting Figure 2).16 Due to the highdimensionality of the experiment, signal overlap was reliablyresolved and 325 cross peaks were automatically picked by theGAPRO algorithm.15† Max Planck Institute for Biophysical Chemistry.§ Max Planck Unit for Structural Molecular Biology.‡ DFG Center for the Molecular Physiology of the Brain.Figure 1. Domain organization (a) and 1H,15N HSQC spectra (b) of thethree Tau isoforms htau23 (no inserts and 3 repeats R1, R3, R4), htau24(no inserts I1, I2 and 4 repeats) and htau40 (441 residues).Published on Web 08/05/201010.1021/ja105657f  2010 American Chemical Society11906 9 J. AM. CHEM. SOC. 2010, 132, 11906–11907Downloaded by MPI BIOPHYSIKALISCHE CHEMIE on August 25, 2015 | http://pubs.acs.org  Publication Date (Web): August 5, 2010 | doi: 10.1021/ja105657fThe 7D HNCO(CA)CBCANH relies on the observation of twosequential HN chemical shifts. Proline and residues following prolinecannot be observed. This is especially problematic in the proline-rich regions P1 and P2 of Tau (22 prolines out of 93 residues). Incontrast, proline succeeding residues are observed in the 5D APSYHACACONH15 resulting in 391 automatically picked cross peakswith HR(i-1), CR(i-1), CO(i-1), N(i), HN(i) chemical shiftinformation (Supporting Information, Table S1).To merge the chemical shift information from the 5D and 7Dexperiments, deviations between the average values of the chemicalshifts in the peak lists of the two spectra are removed. Next identicalspin systems from the two experiments are identified. Two spinsystems are deemed identical if the deviations between the commonchemical shifts H(i)/N(i)/CO(i-1)/Ca(i-1) do not exceed definedthresholds. The threshold values were obtained by a four-dimensional grid search maximizing the number of unique matchesbetween the 7D and 5D experiment. With threshold values of 0.033,0.043, 0.030, and 0.067 ppm for H(i), N(i), CO(i-1), and Ca(i-1),respectively, 294 single matches were obtained. Together withunmatched spin systems from both spectra the merged 7D/5D peaklist contained 416 spin systems.The key step for the automated analysis of the Tau protein is thesequence-specific assignment of the 416 merged spin systems to theprimary sequence. Because of the high dimensionality and the largenumber of spin systems manual assignment is very difficult. In contrast,the high quality of the peak lists makes them suitable for analysis byautomatic assignment methods. Here we extended and improved thealgorithm of the automatic assignment software MARS:17 (i) HN andN connectivity information was implemented and optimized, (ii)random coil chemical shifts were adjusted, and (iii) automatic chemicalshift referencing was introduced.18,19Subjecting the merged 7D/5D peak list of 441-residue htau40to the new MARS algorithm resulted in the automatic assignmentof 92% of assignable residues in less than 3 h (Figure 2a). Forhtau24 (383 residues) and htau23 (352 residues) we also recorded5D HACACONH and 7D HNCO(CA)CBCANH experiments andsubjected them to automatic peak picking, merging of spin systems,and assignment: 95% and 97% of assignable residues were assigned,respectively (Supporting Information, Table S1).In addition, we tested if we can extend the “divide-and-conquer”strategy, which was previously used successfully for manual assign-ment,8 to automatic assignment. To this end, we automatically mergedthe assigned 7D/5D peak list of htau23 with the unassigned 7D/5Dpeak list of htau40, fixed for single matches (see above) the assignmentof peaks of htau40 to those obtained for htau23, and asked MARS tocomplete the assignment of the remaining peaks of htau40. Thisincreased the assignment of htau40 to 93%. Comparison with thepreviously established manual assignment of htau40 at 5 °C, pH 6.8,shows a nearly perfect match (Supporting Figure 3). The few missingassignments are primarily located in the proline-rich region P1 (Figure2a), especially for residues in between two prolines. If required, thesecan be assigned using a 3D HACANNH or a 3D HACAN experiment,which does not rely on detection of amide protons (SupportingFigure 4).20In summary, we demonstrated that the backbone resonanceassignment of very large disordered proteins can be determined byautomatic assignment within 5 days of measurement time and afew hours of calculation reducing the overall analysis time by morethan an order of magnitude. In addition, CO and C chemical shiftsare obtained, which are difficult to obtain from a manual analysisdue to signal overlap, but which are important for analysis ofstructural propensities. The assignment of the three differentisoforms of Tau established in this study at 25 °C allowsinvestigation of isoform specific differences in the structure,microtubule binding, and aggregation of Tau. 15N chemical shiftdifferences between Tau isoforms are not restricted to the segmentborders, providing evidence for the global folding of Tau in solution(Figure 2b and Supporting Figure 5).Acknowledgment. We thank S. Hiller, C. Griesinger, R. Bajaj,the VW foundation, the Max Planck Society, and the DFG(Heisenberg scholarship ZW 71/2-2 and 3-2 to M.Z.) for support.Supporting Information Available: Assignment statistics andchemical shift differences for Tau isoforms. This material is availablefree of charge via the Internet at http://pubs.acs.org.References(1) Dunker, A. K.; Silman, I.; Uversky, V. N.; Sussman, J. L. Curr. Opin.Struct. Biol. 2008, 18, 756–764.(2) Fink, A. L. Curr. Opin. Struct. Biol. 2005, 15, 35–41.(3) Dyson, H. J.; Wright, P. E. Nat. ReV. Mol. Cell Biol. 2005, 6, 197–208.(4) Braak, H.; Braak, E. Acta Neuropathol. (Berlin) 1991, 82, 239–259.(5) Dyson, H. J.; Wright, P. E. Chem. ReV. 2004, 104, 3607–3622.(6) Perez, Y.; Gairi, M.; Pons, M.; Bernado, P. J. Mol. Biol. 2009, 391, 136.(7) Csizmok, V.; Felli, I. C.; Tompa, P.; Banci, L.; Bertini, I. J. Am. Chem.Soc. 2008, 130, 16873–16879.(8) Mukrasch, M. D.; Bibow, S.; Korukottu, J.; Jeganathan, S.; Biernat, J.;Griesinger, C.; Mandelkow, E.; Zweckstetter, M. PLoS Biol. 2009, 7, e34.(9) Smet, C.; Leroy, A.; Sillen, A.; Wieruszeski, J. M.; Landrieu, I.; Lippens,G. ChemBioChem 2004, 5, 1639–1646.(10) Bermel, W.; Bertini, I.; Felli, I. C.; Lee, Y. M.; Luchinat, C.; Pierattelli,R. J. Am. Chem. Soc. 2006, 128, 3918–3919.(11) Mantylahti, S.; Aitio, O.; Hellman, M.; Permi, P. J. Biomol. NMR 2010,47, 171–181.(12) Panchal, S. C.; Bhavesh, N. S.; Hosur, R. V. J. Biomol. NMR 2001, 20,135–147.(13) Zweckstetter, M.; Bax, A. J. Am. Chem. Soc. 2001, 123, 9490–9491.(14) Atreya, H. S.; Eletsky, A.; Szyperski, T. J. Am. Chem. Soc. 2005, 127,4554–4555.(15) Hiller, S.; Fiorito, F.; Wuthrich, K.; Wider, G. Proc. Natl. Acad. Sci. U.S.A.2005, 102, 10876–10881.(16) Hiller, S.; Wasmer, C.; Wider, G.; Wuthrich, K. J. Am. Chem. Soc. 2007,129, 10823–10828.(17) Jung, Y. S.; Zweckstetter, M. J. Biomol. NMR 2004, 30, 11–23.(18) Schwarzinger, S.; Kroon, G. J.; Foss, T. R.; Chung, J.; Wright, P. E.; Dyson,H. J. J. Am. Chem. Soc. 2001, 123, 2970–2978.(19) Wang, L.; Eghbalnia, H. R.; Bahrami, A.; Markley, J. L. J. Biomol. NMR2005, 32, 13–22.(20) Kanelis, V.; Donaldson, L.; Muhandiram, D. R.; Rotin, D.; Forman-Kay,J. D.; Kay, L. E. J. Biomol. NMR 2000, 16, 253–259.JA105657FFigure 2. (a) Automatically obtained assignment of Tau isoforms. Black,gray, and white boxes mark assigned, tentatively assigned, and unassignedresidues, respectively. Proline chemical shifts were obtained from the 5DAPSY from succeeding residues. (b) 15N chemical shift differences betweenhtau40 and htau23. (--) root-mean-square deviation in 15N chemical shiftsbetween 7D and 5D APSY.J. AM. CHEM. SOC. 9 VOL. 132, NO. 34, 2010 11907C O M M U N I C A T I O N SDownloaded by MPI BIOPHYSIKALISCHE CHEMIE on August 25, 2015 | http://pubs.acs.org  Publication Date (Web): August 5, 2010 | doi: 10.1021/ja105657f

Automatic assignment of the intrinsically disordered protein Tau with 441-residues.

http://hdl.handle.net/11858/00-001M-0000-0028-3B10-4

Automatic assignment of the intrinsically disordered protein Tau with 441-residues.

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