- Email: [email protected]
Amphipathic helical behavior of the third repeat fragment in the tau microtubule-binding domain, studied by 1H NMR spectroscopy
BBRC Biochemical and Biophysical Research Communications 294 (2002) 210–214 www.academicpress.com
Amphipathic helical behavior of the third repeat fragment in the tau microtubule-binding domain, studied by 1H NMR spectroscopyq Katsuhiko Minoura,a Koji Tomoo,a Toshimasa Ishida,a,* Hiroshi Hasegawa,b Masahiro Sasaki,b and Taizo Taniguchib,1 b
a Osaka University of Pharmaceutical Sciences, 4-20-1 Nasahara, Takatsuki, Osaka 569-1094, Japan Hyogo Institute for Aging Brain and Cognitive Disorders, 520 Saisho-ko, Himeji, Hyogo 670-0981, Japan
Received 23 April 2002
Abstract The third repeat fragment (3MBD, 31 residues) in the four-repeat microtubule-binding domain of water-soluble tau protein has been considered to be responsible for the formation of the neuropathological filament. To clarify the structural requisite of 3MBD for the filamentous assembly, the solution structures in water and trifluoroethanol (TFE) were investigated by a combination of twodimensional 1 H-NMR measurements and molecular modeling calculations. All protons were assigned by various 2D NMR spectral measurements. The NOE patterns characteristic to the typical helical structure were observed in TFE solution, as was expected from the CD spectra. Using 273 NOE and 23 3 JNHCaH data, possible 3D structures were generated by the dynamical simulated annealing method. The constructed NMR conformers showed that the N-terminal Val1–Lys6 and Leu10–Leu20 fragments form the wellrefined extended and a-helical structures, respectively, whereas the C-terminal moiety is highly flexible. Interestingly, the helical structure showed amphipathic distribution of the respective side chains. This amphipathic behavior of the 3MBD structure would be necessary for self-associating into a helical filament of the tau MBD domain, because such a filament is stabilized by the alternating hydrophilic and hydrophobic interactions between the 3MBD fragments. Ó 2002 Elsevier Science (USA). All rights reserved. Keywords: Tau protein; Microtubule-binding domain; Self-assembly; Helical structure; Amphipathic structure; NMR
Microtubules (MTs) play an important role in the maintenance of cell shape, cell division, axonal transport, secretion, and receptor activity [1]. Microtubuleassociated proteins (MAPs) are believed to be important for MT formation and stabilization [2]. Tau protein, one of the neuronal MAPs in mammalian brain, binds to MTs through the MT-binding domains of three or four
q Abbreviations: MT, microtubule; MAP, microtubule-associated protein; PHF, paired helical filament; MBD, microtubule-binding domain; CD, circular dichroism; DQF-COSY, double quantum filtered chemical-shift correlated spectroscopy; TOCSY, total correlation spectroscopy; NOESY, nuclear Overhauser effect spectroscopy; SA, simulated annealing; NOE, nuclear Overhauser effect; RMSD, root mean square deviation. * Corresponding author. Fax: +81-726-30-1068. E-mail addresses: [email protected] (T. Ishida), t-taizo@ mx6.freecom.ne.jp (T. Taniguchi). 1 Also corresponding author. Present address: Biosignal Research Center, Kobe University, 1-1 Rokkodai-cho, Nada, Kobe 657-8501, Japan. Fax: +81-78-803-5971.
repeated sequences located in the C-terminal half (Fig. 1) [3,4]. Interest on this protein has considerably increased because it is the major component of the pathological lesion that is characteristic of Alzheimer’s disease and other diseases. In these diseases, the tau proteins have been shown to self-aggregate into waterinsoluble structures called paired helical filaments (PHFs) [5,6]. Since these aggregates are thought to be toxic to neurons by obstructing the cell interior, it is of importance to find a method for preventing such pathological aggregation. Regarding the tertiary structure of tau protein, a definite conclusion has not yet been drawn because of its flexible behavior and the structural details of tau selfassembly are far from being completely understood. Concerning the self-assembly, it has been believed that the core structure of PHF is mainly built by the three- or four-repeat microtubule-binding domain (MBD) [7] which promotes the tau assembly in vitro [8]. Therefore, it appears important to examine the structural feature of
0006-291X/02/$ - see front matter Ó 2002 Elsevier Science (USA). All rights reserved. PII: S 0 0 0 6 - 2 9 1 X ( 0 2 ) 0 0 4 5 7 - 6
K. Minoura et al. / Biochemical and Biophysical Research Communications 294 (2002) 210–214
Fig. 1. Schematic diagram of the entire human tau protein (a) and the third amino acid sequence (Val1–Gln31) in the four-repeat MBD moiety. (b) The regions from the first to the fourth MBD repeat in (a) are represented by 1–4MBD, respectively. The numbering of the amino acid residues refers to the longest isoform of human tau (441 residues).
MBD to obtain some clue for understanding the PHF formation. Recently, it was reported [9] that the VQIVYK local sequence of the third repeat in MBD (3MBD in Fig. 1) plays an important role in the assembly of tau protein into Alzheimer PHF. Therefore, in this paper, we report the conformational feature of 3MBD, studied by CD and 1 H-NMR methods.
Materials and methods Peptide. The 31-residue peptide of 3MBD, corresponding to the third repeat segment 306–336 of full-length human tau protein, was synthesized by American Peptide Company (CA, USA), from which synthetic details can be obtained upon request. 3MBD was characterized by mass spectrometry (MALDI-TOF Mþ Na ¼ 3270:38 amu versus theoretical molecular weight ¼ 3247.8 amu) and was pure to >95:0% as assessed by reverse-phase HPLC (column: Vydac C-18; eluents: A ¼ H2 O, 0.1% TFA, B ¼ CH3 CN, 0.1% TFA; linear gradient, 10–40% of B%; retention time ¼ 13.367 min). The sample (including TFA as counterion) was obtained in the form of lyophilized powder. CD measurements. Sample solutions of 3MBD were prepared using water or trifluoroethanol (TFE): 2:0 102 mM in water and 4:0 102 mM in TFE. All measurements at 25 °C were conducted with a JASCO J-820 spectrometer in a cuvette with 2 mm path length. For each experiment under N2 gas flow, measurement from 190 to 260 nm was repeated eight times and the results were summed up. Then, molar ellipticity was determined after normalizing the protein concentrations. The same experiments were performed at least three times using newly prepared samples and their averaged values are given in this paper. Data were expressed in terms of [h], the molar ellipticity, in units of deg cm2 dmol1 . NMR measurements. The peptide was used without further purification by dissolving in TFE–d2 to prepare the sample solution. 1 HNMR spectra were recorded on a Varian unity INOVA500 spectrometer with a variable temperature-control unit. 1 H chemical shifts were referenced to 0 ppm for TSP. From the comparison of respective NMR spectra under different pHs and temperatures, finally the conditions for conventional NMR measurements were determined as follows: concentration ¼ 2 mM, temperature ¼ 298 K, unless otherwise noted, and pH ¼ 3:9 (pH > 5:0 will decrease the solubility of the peptide in solution). The pH value was adjusted by adding HCl or NaOH. For concentration-dependence experiments, the chemical shift of each NH proton was measured under three different concentrations (0.5, 1.0, and 2.0 mM), because of the solubility problem. For temperature-dependence experiments, the chemical shift of each NH proton was measured in the range of 20–60 °C (10 °C intervals). The 2D DQF-COSY, TOCSY, and NOESY spectra were acquired in the
211
phase-sensitive mode using standard pulse programs available in the Varian software library. To trace direct single- and multiple-relayed through-bond connectivities, successively, TOCSY spectra were recorded at mixing times of 40 and 100 ms. The NOESY spectra were also measured at mixing times of 100, 200, and 300 ms. Assuming the same correlation time for all the protons, the offset dependence of the NOESY cross peaks was used for the estimation of proton–proton distances. The NOE intensities were classified into three groups (strong, medium, and weak). On the other hand, the vicinal coupling constants obtained from DQF-COSY measurements were used to estimate possible torsion angles: 3 JHNCaH ¼ 1:9 1:4 cos h þ 6:4 cos2 h, where / ¼ jh 60j° for / torsion angle around C0i1 ANi ACai AC0i bond sequence [10]. Computational molecular modeling. Various 3D structures that satisfy the NOE distance and J torsion angle constraints of intramolecular proton pairs were constructed by dynamic simulated annealing (SA) calculations [11,12] using the CNS [13] program. The protocol for the SA calculation is as follows. After the randomization of the peptide into the extended strands corresponding to each disjoint molecular entity, the protocol consists of reading various data structures and the initialization of statistical analysis of average property. The constructed structures are then annealed for 15 ps at 50,000 K and cooled to 300 K with a slope of 250 K/step for 10 ps. Finally, the minimization of the peptide over 5000 steps is continued. The constraints for distance and torsion angle were used as the harmonic potential function. As input data for the distance constraint, the proton–proton pairs were classified into three distance groups according to the NOE intensities: ), medium (1.8–4.0 A ), and weak (1.8–5.0 A ). The strong (1.8–3.0 A torsional constraint was applied for torsion / angle, i.e., 120 40° for 3 JHNCaH > 8 Hz, 110 50° for 3 JHNCaH > 7 Hz, 75 25° for 3 JHNCaH < 6 Hz, and 100 60° for the others. RMSD analyses of energy minimized structures were carried out using the program MOLMOL [14].
Results and discussion To study the overall conformation of 3MBD, the CD spectra were measured in water (pH 4.2 and 7.0) and TFE (pH 3.9 and 7.2) (Fig. 2). Although the quantification of CD spectra in terms of secondary structure components is often unreliable, the general shapes of the
Fig. 2. CD spectra of 3MBD in water and TFE solutions.
212
K. Minoura et al. / Biochemical and Biophysical Research Communications 294 (2002) 210–214
Fig. 3. Diagram of NOE connectivity between neighboring (daNði;iþ1Þ , dbNði;iþ1Þ , and dNNði;iþ1Þ ) protons and J-coupling constants (3 JHNACaH ). The strength of the observed NOE is represented by the thickness of the respective bars. Residues with 3 JHNACaH < 6 Hz are indicated by arrows.
Table 1 Structural statistics for 20 stable structures of 3MBD domain Average values (esd) RMS deviation (N, Ca, C0 )a ) RMS deviation from NOE (A NOE violations > 0:10 A
0.6 (2) 0.008 (1) 3.2 (4)
Energies (kcal/mol) Overall NOE Angle Bond Improper van der Waals
85 (2) 32 (1) 30 (1) 7.3 (3) 6.4 (1) 8 (1)
a
Calculated over residues 10–20.
spectra are significant because they reveal gross conformational states. The CD spectra of 3MBD in TFE are indicative of an a-helical structure characterized by two negative peaks around 209 and 222 nm, the conformation of which is slightly affected by pH variation, whereas those in water showed a random conformation characterized by a negative peak around 197 nm. This result indicates the solvent-dependent behavior of 3MBD conformation. To determine the 3MBD structure in TFE solvent, the solution conformation was analyzed by a combination of 1 H-NMR spectroscopy and molecular modeling calculation. Proton peak assignments were performed by a combination of (a) the connectivity information via scalar coupling in phase-sensitive TOCSY experiments and (b) the sequential NOE networks along the peptide backbone protons. The NOESY spectra were then measured to observe short-, medium-, and long-range proton–proton connectivities along the peptide backbone. The results are summarized in Fig. 3. The trans orientation around the Lys6–Pro7 and Lys26–Pro27 x bonds was determined from the strong NOEs of CaH (Lys6)–CdH (Pro7) and CaH (Lys26)–CdH (Pro27) proton pairs. The NOESY cross-peak pattern [15] among neighboring protons suggested the existence of the b-sheet structure for Gln2–Lys6 sequence and the ahelical structure of Val8–Leu20 sequence. Using 273
Fig. 4. Stereoscopic superposition of the 20 most stable conformers of 3MBD. Each conformer is projected to superimpose the Leu10–Leu20 sequence.
K. Minoura et al. / Biochemical and Biophysical Research Communications 294 (2002) 210–214
Fig. 5. Helical wheel drawing of the Leu10–Leu20 sequence of the most stable conformer of 3MBD, viewed down from the N-terminal side.
213
has been reported to be necessary for the assembly of tau protein into Alzheimer PHFs [9] and the extended structure of this sequence may be important as a trigger for the self-aggregation. On the other hand, the sequence of Leu10–Leu20 forms a well-defined a-helical ). Instructure (RMS deviation of backbone is 0.55 A terestingly, the helix wheel drawing of this sequence shows the amphipathic distribution of the respective amino acid residues (Fig. 5). The hydrophobic residues of Leu10 and Val13 and the hydrophilic residues of Ser11, Thr14, Ser15, and Ser19 are arranged on the two sides of the helix axis, respectively, and the polar residues of Cys17, Lys12, and Lys16 are located at the interface between the two sides. Although the biological significance of such an orientation is unknown at present, the following consideration is possible for the selfassembly of MBD. Fig. 6 shows a possible assembly model via the helical structure of 3MBD, i.e., the dimer formation due to the hydrophilic interactions and then the molecular aggregation of these dimer structures due to the hydrophobic interactions. In this model, the piling axis of the assembly is at right angles to each MBD molecule; a similar model has already been proposed by Friedhoff et al. [16]. In conclusion, the amphipathic helical structure of 3MBD in TFE was first clarified by the present work and a possible self-assembly model of the MBD domain in tau protein is proposed. The results may be useful for understanding the biological events occurring at the initial step of abnormal filament aggregation of tau protein in Alzheimer’s disease.
References
Fig. 6. A possible assembly model of 3MBD. Self-assembly is performed via (a) antiparallel dimer formation due to the hydrophilic interactions between polar amino acid residues and (b) molecular assembly of dimer structures due to the hydrophobic interactions between nonpolar amino acid residues.
NOE constraints for proton–proton distances and 23 3 JHNCaH constraints for / torsion angles, the construction of possible conformers was attempted by dynamic SA calculation. One hundred 3D structures of 3MBD were constructed and the statistics for these structures are summarized in Table 1. The superposition of the backbone structures of the 20 most stable conformers is shown in Fig. 4. The results indicate that the N-terminal Val1–Lys6 and Leu10–Leu20 fragments form the extended and a-helical structures, respectively, whereas the C-terminal moiety is flexible and does not take any definite 3D structure. The VQIVYK sequence in 3MBD
[1] R.D. Burgoyne, The Neuronal Cytoskeleton, Wiley-Liss, New York, 1991. [2] M. Goedert, R. Jakes, M.G. Spillantini, R.A. Crowther, in: J. Hyams, C. Lloyd (Eds.), Microtubules, Wiley-Liss, New York, 1994, pp. 183–200. [3] L. Varani, M. Hasegawa, M.G. Spillantini, M.J. Smith, J.R. Murrell, B. Ghetti, A. Klug, M. Goedert, G. Varani, Structure of tau exon 10 splicing regulatory element RNA and destabilization by mutations of frontotemporal dementia and parkinsonism linked to chromosome 17, Proc. Natl. Acad. Sci. USA 96 (1999) 8229–8234. [4] P. Friedhoff, M. von Bergen, E.-M. Mandelkow, E. Mandelkow, Structure of tau protein and assembly into paired helical filaments, Biochim. Biophys. Acta 1502 (2000) 122–132. [5] M. Goedert, M.G. Spillantini, Tau mutations in frontotemporal dementia FTDP-17 and their relevance for Alzheimer’s disease, Biochim. Biophys. Acta 1502 (2000) 110–121. [6] M. Hasegawa, M.J. Smith, M. Goedert, Tau proteins with FTDP17 mutations have a reduced ability to promote microtubule assembly, FEBS Lett. 437 (1998) 207–210. [7] P. Friedhoff, M. von Bergen, E.-M. Mandelkow, E. Mandelkow, A nucleated assembly mechanism of Alzheimer paired helical filaments, Proc. Natl. Acad. Sci. USA 95 (1998) 15712– 15717.
214
K. Minoura et al. / Biochemical and Biophysical Research Communications 294 (2002) 210–214
[8] H. Wille, G. Drewes, J. Biernat, E.-M. Mandelkow, E. Mandelkow, Alzheimer-like paired helical filaments and antiparallel dimers formed from microtubule-associated protein tau in vitro, J. Cell Biol. 118 (1992) 573–584. [9] M. Von Bergen, P. Friedhoff, J. Biernat, J. Heberle, E.M. Mandelkow, E. Mandelkow, Assembly of tau protein into Alzheimer paired helical filaments depends on a local sequence motif ((306)VQIVYK(311)) forming beta structure, Proc. Natl. Acad. Sci. USA 97 (2000) 5129–5134. [10] V.F. Bystrov, Spin-spin coupling and the conformational states of peptide systems, Prog. Nucl. Magn. Reson. Spectrosc. 10 (1976) 41–81. [11] G.M. Clore, M. Nigles, D.K. Sukumaran, A.T. Bruenger, M. Karplus, A.M. Gronenborn, The three-dimensional structure of a1-purothionin in solution: combined use of nuclear magnetic resonance, distance geometry and restrained molecular dynamics, EMBO J. 5 (1986) 2729–2735. [12] M. Nilges, G.M. Clore, A.M. Gronenborn, Determination of three-dimensional structures of proteins from interproton distance
[13]
[14]
[15]
[16]
data by hybrid distance geometry-dynamical simulated annealing calculations, FEBS Lett. 229 (1988) 317–324. A.T. Brunger, P.D. Adams, G.M. Clore, W.L. DeLano, P. Gros, R.W. Grosse-Kunstleve, J.S. Jiang, J. Kuszewski, M. Nilges, N.S. Pannu, R.J. Read, L.M. Rice, T. Simonson, G.L. Warren, Crystallography & NMR system: a new software suite for macromolecular structure determination, Acta Crystallogr. D 54 (1998) 905–921. R. Koradi, M. Billeter, K. Wuthrich, MOLMOL: a program for display and analysis of macromolecular structures, J. Mol. Graph. 14 (1996) 51–55. G. Wagner, D. Neuhaus, E. Worgotter, M. Vasak, J.H. Kagi, K. Wuthrich, Nuclear magnetic resonance identification of ‘‘halfturn’’ and 3(10)-helix secondary structure in rabbit liver metallothionein-2, J. Mol. Biol. 187 (1986) 131–135. P. Friedhoff, M. von Bergen, E.-M. Mandelkow, E. Mandelkow, A nucleated assembly mechanism of Alzheimer paired helical filaments, Proc. Natl. Acad. Sci. USA 95 (1998) 15712– 15717.
Amphipathic helical behavior of the third repeat fragment in the tau microtubule-binding domain, studied by 1H NMR spectroscopyq Katsuhiko Minoura,a Koji Tomoo,a Toshimasa Ishida,a,* Hiroshi Hasegawa,b Masahiro Sasaki,b and Taizo Taniguchib,1 b
a Osaka University of Pharmaceutical Sciences, 4-20-1 Nasahara, Takatsuki, Osaka 569-1094, Japan Hyogo Institute for Aging Brain and Cognitive Disorders, 520 Saisho-ko, Himeji, Hyogo 670-0981, Japan
Received 23 April 2002
Abstract The third repeat fragment (3MBD, 31 residues) in the four-repeat microtubule-binding domain of water-soluble tau protein has been considered to be responsible for the formation of the neuropathological filament. To clarify the structural requisite of 3MBD for the filamentous assembly, the solution structures in water and trifluoroethanol (TFE) were investigated by a combination of twodimensional 1 H-NMR measurements and molecular modeling calculations. All protons were assigned by various 2D NMR spectral measurements. The NOE patterns characteristic to the typical helical structure were observed in TFE solution, as was expected from the CD spectra. Using 273 NOE and 23 3 JNHCaH data, possible 3D structures were generated by the dynamical simulated annealing method. The constructed NMR conformers showed that the N-terminal Val1–Lys6 and Leu10–Leu20 fragments form the wellrefined extended and a-helical structures, respectively, whereas the C-terminal moiety is highly flexible. Interestingly, the helical structure showed amphipathic distribution of the respective side chains. This amphipathic behavior of the 3MBD structure would be necessary for self-associating into a helical filament of the tau MBD domain, because such a filament is stabilized by the alternating hydrophilic and hydrophobic interactions between the 3MBD fragments. Ó 2002 Elsevier Science (USA). All rights reserved. Keywords: Tau protein; Microtubule-binding domain; Self-assembly; Helical structure; Amphipathic structure; NMR
Microtubules (MTs) play an important role in the maintenance of cell shape, cell division, axonal transport, secretion, and receptor activity [1]. Microtubuleassociated proteins (MAPs) are believed to be important for MT formation and stabilization [2]. Tau protein, one of the neuronal MAPs in mammalian brain, binds to MTs through the MT-binding domains of three or four
q Abbreviations: MT, microtubule; MAP, microtubule-associated protein; PHF, paired helical filament; MBD, microtubule-binding domain; CD, circular dichroism; DQF-COSY, double quantum filtered chemical-shift correlated spectroscopy; TOCSY, total correlation spectroscopy; NOESY, nuclear Overhauser effect spectroscopy; SA, simulated annealing; NOE, nuclear Overhauser effect; RMSD, root mean square deviation. * Corresponding author. Fax: +81-726-30-1068. E-mail addresses: [email protected] (T. Ishida), t-taizo@ mx6.freecom.ne.jp (T. Taniguchi). 1 Also corresponding author. Present address: Biosignal Research Center, Kobe University, 1-1 Rokkodai-cho, Nada, Kobe 657-8501, Japan. Fax: +81-78-803-5971.
repeated sequences located in the C-terminal half (Fig. 1) [3,4]. Interest on this protein has considerably increased because it is the major component of the pathological lesion that is characteristic of Alzheimer’s disease and other diseases. In these diseases, the tau proteins have been shown to self-aggregate into waterinsoluble structures called paired helical filaments (PHFs) [5,6]. Since these aggregates are thought to be toxic to neurons by obstructing the cell interior, it is of importance to find a method for preventing such pathological aggregation. Regarding the tertiary structure of tau protein, a definite conclusion has not yet been drawn because of its flexible behavior and the structural details of tau selfassembly are far from being completely understood. Concerning the self-assembly, it has been believed that the core structure of PHF is mainly built by the three- or four-repeat microtubule-binding domain (MBD) [7] which promotes the tau assembly in vitro [8]. Therefore, it appears important to examine the structural feature of
0006-291X/02/$ - see front matter Ó 2002 Elsevier Science (USA). All rights reserved. PII: S 0 0 0 6 - 2 9 1 X ( 0 2 ) 0 0 4 5 7 - 6
K. Minoura et al. / Biochemical and Biophysical Research Communications 294 (2002) 210–214
Fig. 1. Schematic diagram of the entire human tau protein (a) and the third amino acid sequence (Val1–Gln31) in the four-repeat MBD moiety. (b) The regions from the first to the fourth MBD repeat in (a) are represented by 1–4MBD, respectively. The numbering of the amino acid residues refers to the longest isoform of human tau (441 residues).
MBD to obtain some clue for understanding the PHF formation. Recently, it was reported [9] that the VQIVYK local sequence of the third repeat in MBD (3MBD in Fig. 1) plays an important role in the assembly of tau protein into Alzheimer PHF. Therefore, in this paper, we report the conformational feature of 3MBD, studied by CD and 1 H-NMR methods.
Materials and methods Peptide. The 31-residue peptide of 3MBD, corresponding to the third repeat segment 306–336 of full-length human tau protein, was synthesized by American Peptide Company (CA, USA), from which synthetic details can be obtained upon request. 3MBD was characterized by mass spectrometry (MALDI-TOF Mþ Na ¼ 3270:38 amu versus theoretical molecular weight ¼ 3247.8 amu) and was pure to >95:0% as assessed by reverse-phase HPLC (column: Vydac C-18; eluents: A ¼ H2 O, 0.1% TFA, B ¼ CH3 CN, 0.1% TFA; linear gradient, 10–40% of B%; retention time ¼ 13.367 min). The sample (including TFA as counterion) was obtained in the form of lyophilized powder. CD measurements. Sample solutions of 3MBD were prepared using water or trifluoroethanol (TFE): 2:0 102 mM in water and 4:0 102 mM in TFE. All measurements at 25 °C were conducted with a JASCO J-820 spectrometer in a cuvette with 2 mm path length. For each experiment under N2 gas flow, measurement from 190 to 260 nm was repeated eight times and the results were summed up. Then, molar ellipticity was determined after normalizing the protein concentrations. The same experiments were performed at least three times using newly prepared samples and their averaged values are given in this paper. Data were expressed in terms of [h], the molar ellipticity, in units of deg cm2 dmol1 . NMR measurements. The peptide was used without further purification by dissolving in TFE–d2 to prepare the sample solution. 1 HNMR spectra were recorded on a Varian unity INOVA500 spectrometer with a variable temperature-control unit. 1 H chemical shifts were referenced to 0 ppm for TSP. From the comparison of respective NMR spectra under different pHs and temperatures, finally the conditions for conventional NMR measurements were determined as follows: concentration ¼ 2 mM, temperature ¼ 298 K, unless otherwise noted, and pH ¼ 3:9 (pH > 5:0 will decrease the solubility of the peptide in solution). The pH value was adjusted by adding HCl or NaOH. For concentration-dependence experiments, the chemical shift of each NH proton was measured under three different concentrations (0.5, 1.0, and 2.0 mM), because of the solubility problem. For temperature-dependence experiments, the chemical shift of each NH proton was measured in the range of 20–60 °C (10 °C intervals). The 2D DQF-COSY, TOCSY, and NOESY spectra were acquired in the
211
phase-sensitive mode using standard pulse programs available in the Varian software library. To trace direct single- and multiple-relayed through-bond connectivities, successively, TOCSY spectra were recorded at mixing times of 40 and 100 ms. The NOESY spectra were also measured at mixing times of 100, 200, and 300 ms. Assuming the same correlation time for all the protons, the offset dependence of the NOESY cross peaks was used for the estimation of proton–proton distances. The NOE intensities were classified into three groups (strong, medium, and weak). On the other hand, the vicinal coupling constants obtained from DQF-COSY measurements were used to estimate possible torsion angles: 3 JHNCaH ¼ 1:9 1:4 cos h þ 6:4 cos2 h, where / ¼ jh 60j° for / torsion angle around C0i1 ANi ACai AC0i bond sequence [10]. Computational molecular modeling. Various 3D structures that satisfy the NOE distance and J torsion angle constraints of intramolecular proton pairs were constructed by dynamic simulated annealing (SA) calculations [11,12] using the CNS [13] program. The protocol for the SA calculation is as follows. After the randomization of the peptide into the extended strands corresponding to each disjoint molecular entity, the protocol consists of reading various data structures and the initialization of statistical analysis of average property. The constructed structures are then annealed for 15 ps at 50,000 K and cooled to 300 K with a slope of 250 K/step for 10 ps. Finally, the minimization of the peptide over 5000 steps is continued. The constraints for distance and torsion angle were used as the harmonic potential function. As input data for the distance constraint, the proton–proton pairs were classified into three distance groups according to the NOE intensities: ), medium (1.8–4.0 A ), and weak (1.8–5.0 A ). The strong (1.8–3.0 A torsional constraint was applied for torsion / angle, i.e., 120 40° for 3 JHNCaH > 8 Hz, 110 50° for 3 JHNCaH > 7 Hz, 75 25° for 3 JHNCaH < 6 Hz, and 100 60° for the others. RMSD analyses of energy minimized structures were carried out using the program MOLMOL [14].
Results and discussion To study the overall conformation of 3MBD, the CD spectra were measured in water (pH 4.2 and 7.0) and TFE (pH 3.9 and 7.2) (Fig. 2). Although the quantification of CD spectra in terms of secondary structure components is often unreliable, the general shapes of the
Fig. 2. CD spectra of 3MBD in water and TFE solutions.
212
K. Minoura et al. / Biochemical and Biophysical Research Communications 294 (2002) 210–214
Fig. 3. Diagram of NOE connectivity between neighboring (daNði;iþ1Þ , dbNði;iþ1Þ , and dNNði;iþ1Þ ) protons and J-coupling constants (3 JHNACaH ). The strength of the observed NOE is represented by the thickness of the respective bars. Residues with 3 JHNACaH < 6 Hz are indicated by arrows.
Table 1 Structural statistics for 20 stable structures of 3MBD domain Average values (esd) RMS deviation (N, Ca, C0 )a ) RMS deviation from NOE (A NOE violations > 0:10 A
0.6 (2) 0.008 (1) 3.2 (4)
Energies (kcal/mol) Overall NOE Angle Bond Improper van der Waals
85 (2) 32 (1) 30 (1) 7.3 (3) 6.4 (1) 8 (1)
a
Calculated over residues 10–20.
spectra are significant because they reveal gross conformational states. The CD spectra of 3MBD in TFE are indicative of an a-helical structure characterized by two negative peaks around 209 and 222 nm, the conformation of which is slightly affected by pH variation, whereas those in water showed a random conformation characterized by a negative peak around 197 nm. This result indicates the solvent-dependent behavior of 3MBD conformation. To determine the 3MBD structure in TFE solvent, the solution conformation was analyzed by a combination of 1 H-NMR spectroscopy and molecular modeling calculation. Proton peak assignments were performed by a combination of (a) the connectivity information via scalar coupling in phase-sensitive TOCSY experiments and (b) the sequential NOE networks along the peptide backbone protons. The NOESY spectra were then measured to observe short-, medium-, and long-range proton–proton connectivities along the peptide backbone. The results are summarized in Fig. 3. The trans orientation around the Lys6–Pro7 and Lys26–Pro27 x bonds was determined from the strong NOEs of CaH (Lys6)–CdH (Pro7) and CaH (Lys26)–CdH (Pro27) proton pairs. The NOESY cross-peak pattern [15] among neighboring protons suggested the existence of the b-sheet structure for Gln2–Lys6 sequence and the ahelical structure of Val8–Leu20 sequence. Using 273
Fig. 4. Stereoscopic superposition of the 20 most stable conformers of 3MBD. Each conformer is projected to superimpose the Leu10–Leu20 sequence.
K. Minoura et al. / Biochemical and Biophysical Research Communications 294 (2002) 210–214
Fig. 5. Helical wheel drawing of the Leu10–Leu20 sequence of the most stable conformer of 3MBD, viewed down from the N-terminal side.
213
has been reported to be necessary for the assembly of tau protein into Alzheimer PHFs [9] and the extended structure of this sequence may be important as a trigger for the self-aggregation. On the other hand, the sequence of Leu10–Leu20 forms a well-defined a-helical ). Instructure (RMS deviation of backbone is 0.55 A terestingly, the helix wheel drawing of this sequence shows the amphipathic distribution of the respective amino acid residues (Fig. 5). The hydrophobic residues of Leu10 and Val13 and the hydrophilic residues of Ser11, Thr14, Ser15, and Ser19 are arranged on the two sides of the helix axis, respectively, and the polar residues of Cys17, Lys12, and Lys16 are located at the interface between the two sides. Although the biological significance of such an orientation is unknown at present, the following consideration is possible for the selfassembly of MBD. Fig. 6 shows a possible assembly model via the helical structure of 3MBD, i.e., the dimer formation due to the hydrophilic interactions and then the molecular aggregation of these dimer structures due to the hydrophobic interactions. In this model, the piling axis of the assembly is at right angles to each MBD molecule; a similar model has already been proposed by Friedhoff et al. [16]. In conclusion, the amphipathic helical structure of 3MBD in TFE was first clarified by the present work and a possible self-assembly model of the MBD domain in tau protein is proposed. The results may be useful for understanding the biological events occurring at the initial step of abnormal filament aggregation of tau protein in Alzheimer’s disease.
References
Fig. 6. A possible assembly model of 3MBD. Self-assembly is performed via (a) antiparallel dimer formation due to the hydrophilic interactions between polar amino acid residues and (b) molecular assembly of dimer structures due to the hydrophobic interactions between nonpolar amino acid residues.
NOE constraints for proton–proton distances and 23 3 JHNCaH constraints for / torsion angles, the construction of possible conformers was attempted by dynamic SA calculation. One hundred 3D structures of 3MBD were constructed and the statistics for these structures are summarized in Table 1. The superposition of the backbone structures of the 20 most stable conformers is shown in Fig. 4. The results indicate that the N-terminal Val1–Lys6 and Leu10–Leu20 fragments form the extended and a-helical structures, respectively, whereas the C-terminal moiety is flexible and does not take any definite 3D structure. The VQIVYK sequence in 3MBD
[1] R.D. Burgoyne, The Neuronal Cytoskeleton, Wiley-Liss, New York, 1991. [2] M. Goedert, R. Jakes, M.G. Spillantini, R.A. Crowther, in: J. Hyams, C. Lloyd (Eds.), Microtubules, Wiley-Liss, New York, 1994, pp. 183–200. [3] L. Varani, M. Hasegawa, M.G. Spillantini, M.J. Smith, J.R. Murrell, B. Ghetti, A. Klug, M. Goedert, G. Varani, Structure of tau exon 10 splicing regulatory element RNA and destabilization by mutations of frontotemporal dementia and parkinsonism linked to chromosome 17, Proc. Natl. Acad. Sci. USA 96 (1999) 8229–8234. [4] P. Friedhoff, M. von Bergen, E.-M. Mandelkow, E. Mandelkow, Structure of tau protein and assembly into paired helical filaments, Biochim. Biophys. Acta 1502 (2000) 122–132. [5] M. Goedert, M.G. Spillantini, Tau mutations in frontotemporal dementia FTDP-17 and their relevance for Alzheimer’s disease, Biochim. Biophys. Acta 1502 (2000) 110–121. [6] M. Hasegawa, M.J. Smith, M. Goedert, Tau proteins with FTDP17 mutations have a reduced ability to promote microtubule assembly, FEBS Lett. 437 (1998) 207–210. [7] P. Friedhoff, M. von Bergen, E.-M. Mandelkow, E. Mandelkow, A nucleated assembly mechanism of Alzheimer paired helical filaments, Proc. Natl. Acad. Sci. USA 95 (1998) 15712– 15717.
214
K. Minoura et al. / Biochemical and Biophysical Research Communications 294 (2002) 210–214
[8] H. Wille, G. Drewes, J. Biernat, E.-M. Mandelkow, E. Mandelkow, Alzheimer-like paired helical filaments and antiparallel dimers formed from microtubule-associated protein tau in vitro, J. Cell Biol. 118 (1992) 573–584. [9] M. Von Bergen, P. Friedhoff, J. Biernat, J. Heberle, E.M. Mandelkow, E. Mandelkow, Assembly of tau protein into Alzheimer paired helical filaments depends on a local sequence motif ((306)VQIVYK(311)) forming beta structure, Proc. Natl. Acad. Sci. USA 97 (2000) 5129–5134. [10] V.F. Bystrov, Spin-spin coupling and the conformational states of peptide systems, Prog. Nucl. Magn. Reson. Spectrosc. 10 (1976) 41–81. [11] G.M. Clore, M. Nigles, D.K. Sukumaran, A.T. Bruenger, M. Karplus, A.M. Gronenborn, The three-dimensional structure of a1-purothionin in solution: combined use of nuclear magnetic resonance, distance geometry and restrained molecular dynamics, EMBO J. 5 (1986) 2729–2735. [12] M. Nilges, G.M. Clore, A.M. Gronenborn, Determination of three-dimensional structures of proteins from interproton distance
[13]
[14]
[15]
[16]
data by hybrid distance geometry-dynamical simulated annealing calculations, FEBS Lett. 229 (1988) 317–324. A.T. Brunger, P.D. Adams, G.M. Clore, W.L. DeLano, P. Gros, R.W. Grosse-Kunstleve, J.S. Jiang, J. Kuszewski, M. Nilges, N.S. Pannu, R.J. Read, L.M. Rice, T. Simonson, G.L. Warren, Crystallography & NMR system: a new software suite for macromolecular structure determination, Acta Crystallogr. D 54 (1998) 905–921. R. Koradi, M. Billeter, K. Wuthrich, MOLMOL: a program for display and analysis of macromolecular structures, J. Mol. Graph. 14 (1996) 51–55. G. Wagner, D. Neuhaus, E. Worgotter, M. Vasak, J.H. Kagi, K. Wuthrich, Nuclear magnetic resonance identification of ‘‘halfturn’’ and 3(10)-helix secondary structure in rabbit liver metallothionein-2, J. Mol. Biol. 187 (1986) 131–135. P. Friedhoff, M. von Bergen, E.-M. Mandelkow, E. Mandelkow, A nucleated assembly mechanism of Alzheimer paired helical filaments, Proc. Natl. Acad. Sci. USA 95 (1998) 15712– 15717.