Phosphorylation modulates the local conformation and self-aggregation ability of a peptide from the fourth tau microtubule-binding repeat Jin-Tang Du 1 , Chun-Hui Yu 1 , Lian-Xiu Zhou 1 , Wei-Hui Wu 1 , Peng Lei 1 , Yong Li 1 , Yu-Fen Zhao 1 , Hiroshi Nakanishi 2 and Yan-Mei Li 1 1 Key Laboratory of Bioorganic Phosphorus Chemistry and Chemical Biology (Ministry of Education), Tsinghua University, Beijing, China 2 Research Institute of Instrumentation Frontier, National Institute of Advanced Industrial Science and Technology, Ibaraki, Japan Post-translational phosphorylation serves as a control mechanism in a myriad of cellular processes including metabolic pathway regulation, extracellular signal transduction, ion channel regulation and cell-cycle pro- gression [1]. In other cases, abnormal phosphorylation may be harmful; for example, hyperphosphorylation causes the aggregation of microtubule-associated pro- tein tau, which is implicated in Alzheimer’s disease (AD) [2,3]. AD is the main form of dementia in today’s ageing population [2]. It is characterized by the presence of two aberrant structures, senile plaques and neurofibril- lary tangles. The main components of neurofibrillary tangles are paired helical filaments (PHFs) [4], which are mainly comprised of the protein tau in an abnor- mally phosphorylated form [3]. Tau protein, whose main function is to stimulate and stabilize microtubule assembly from tubulin subunits, is abundant in both the central and peripheral nervous systems [5]. Tau stabilizes microtubules and regulates the transport of vesicles or organelles along them, it supports the outgrowth of axons and serves as an anchor for enzymes [6]. Tau binds to microtubules via the micro- tubule-binding domain, which contains four copies of a highly conserved 18-amino acid repeat, namely, R1, R2, R3 and R4, each of which is separated from another repeat by less conserved 13- or 14-amino acid inter-repeat domains [7]. Although tau protein is water soluble and shows little tendency to aggregate under physiological conditions, it dissociates from microtu- bules and aggregates into PHFs in the brains of AD patients [8–12]. Functionally, tau binds to tubulin, whereas PHF-tau does not [10–15]. Because this aggre- gation leads to toxicity in neurons due to damage to Keywords aggregation; Alzheimer’s disease; microtubule-binding repeat; phosphorylation; tau Correspondence Y M. Li, Key Laboratory of Bioorganic Phosphorus Chemistry & Chemical Biology (Ministry of Education), Department of Chemistry, Tsinghua University, Beijing 100084, China Fax: +86 10 6278 1695 Tel: +86 10 6279 6197 E-mail: liym@mail.tsinghua.edu.cn (Received 27 May 2007, revised 24 July 2007, accepted 30 July 2007) doi:10.1111/j.1742-4658.2007.06018.x Phosphorylation of tau protein modulates both its physiological role and its aggregation into paired helical fragments, as observed in Alzheimer’s diseased neurons. It is of fundamental importance to study paired helical fragment formation and its modulation by phosphorylation. This study focused on the fourth microtubule-binding repeat of tau, encompassing an abnormal phosphorylation site, Ser356. The aggregation propensities of this repeat peptide and its corresponding phosphorylated form were investi- gated using turbidity, thioflavin T fluorescence and electron microscopy. There is evidence for a conformational change in the fourth microtubule- binding repeat of tau peptide upon phosphorylation, as well as changes in aggregation activity. Although both tau peptides have the ability to aggre- gate, this is weaker in the phosphorylated peptide. This study reveals that both tau peptides are capable of self-aggregation and that phosphorylation at Ser356 can modulate this process. Abbreviations AD, Alzheimer’s disease; PHF, paired helical filament; ThT, thioflavin T. 5012 FEBS Journal 274 (2007) 5012–5020 ª 2007 The Authors Journal compilation ª 2007 FEBS the cell interior, it is important to clarify the mecha- nism of aggregation of tau protein and develop ways to prevent this pathological assembly. The microtubule-binding domain, located in the C-terminal of tau protein, has been reported to assume the core structure of PHFs and promote tau aggrega- tion in vitro [16–18]. We previously studied the metal- binding properties and the effects of phosphorylation on tau protein fragments. Several tentative explana- tions for PHF formation have been proposed [19–21]. It is also observed that the second (R2) and third (R3) microtubule-binding repeats can aggregate with the help of heparin [22]. However, little such data concern- ing the contribution of the R4 repeat to the formation of PHFs and the modulation by phosphorylation have been reported. Here, we investigate the aggregation propensity of the R4 repeat using turbidity measurements, thiofla- vin T (ThT) fluorescence and electron microscopy. Tur- bidity measurements are an excellent, widely adopted method of quantifying aggregation in solution [23,24]. In addition, it has been suggested that phosphorylation of some specific tau sites may be a prerequisite for aggregation [25,26]. Ser356, which is located in the R4 repeat, is one likely abnormal phosphorylation sites [27]. It is not clear how abnormal phosphorylation of tau protein modulates aggregation. In vitro, the stimu- latory effects of phosphorylation on the aggregation of tau have been reported [28]. However, different phos- phorylation sites may have different effects on filament formation, and it is advantageous to study the effect of only one confined phosphorylation site on a tau pep- tide. However, analysis of the effect of phosphorylation at defined sites is hampered by the low specificity of protein kinases and the highly dynamic turnover of phosphorylation in vivo. Site-directed mutagenesis, which converts serine and threonine to aspartic acid and glutamic acid, has been used to imitate phosphory- lation [29]. In our study, synthetic phosphopeptide was used and the effect of phosphorylation on the tau repeat fragment assembly was also studied. Recently, Wang et al. showed that AD P-tau dephosphorylated by protein phosphatase did not aggre- gate into filaments, whereas several protein kinases and their combinations can abnormally hyperphos- phate protein phosphatase dephosphorylation of AD P-tau and induce its self-aggregation into PHF similar to those seen in AD. It is, thus, important to learn how phosphorylation modulates the self-aggregation of tau. This study focused on the aggregation propensity of the fourth microtubule-binding repeat of tau peptide in its unphosphorylated (R4) and also phosphorylated (pR4) form [30], to try and explain how phosphoryla- tion modulates the process of aggregation at the molecular level. Peptide R4 and phosphopeptide pR4 relating to the human tau protein (Table 1) were syn- thesized according to a solid-phase synthetic strategy. Phosphopeptide pR4 was phosphorylated at Ser356. The structural differences between phosphopeptide and nonphosphopeptide were analysed using CD and high- resolution NMR spectroscopy. The aggregation behav- ior of peptide R4 and phosphopeptide pR4 and the structural differences between them were then exam- ined using turbidity, ThT fluorescence and electron microscopy. The results from turbidity measurements, ThT fluorescence and electron microscopy show that the R4 repeat and its phosphorylated form pR4 are capable of self-aggregation. It is proposed that this repeat plays an important role in the aggregation of tau protein and phosphorylation is able to modulate the process of aggregation. Results Phosphorylation of Ser356-induced conforma- tional change in peptide R4 To investigate the effect of phosphorylation at Ser356 on the native structure of peptide R4, both CD and NMR spectroscopy were performed. In NMR spectroscopy, TOCSY and NOESY spectra of the two peptides were recorded and compared. Changes in the backbone NH and aH chemical shifts upon phosphorylation (d phosphorylated –d nonphosphorylated ) were shown for each residue. A comparison of the chemical shifts of NH and aH between the non- phosphorylated and phosphorylated peptides is sum- marized in Fig. 1. The chemical shift deviations of NH and aH reflect changes in the electrostatic state and molecular structure. Upon phosphorylation at Ser356, the largest proton chemical shift deviation of NH and aH was observed for Ser356 (downfield 0.41 p.p.m. for Table 1. Synthetic peptides corresponding to the repeat domain of the human tau441 sequence. p, phosphorylation. Tau peptide Amino acid sequence Repeat number R4 VQSKIGSLDNITHVPGGG 350–367 ⁄ fourth repeat pR4 VQSKIGpSLDNITHVPGGG 350–367 ⁄ fourth repeat J T. Du et al. Modulation of tau R4 peptide by phosphorylation FEBS Journal 274 (2007) 5012–5020 ª 2007 The Authors Journal compilation ª 2007 FEBS 5013 NH and 0.10 p.p.m. for aH). In general, the chemical shift of NH deviates was more than that of aH upon phosphorylation. Notable chemical shift deviation of NH and aH occurs mainly at the phosphorylation site and sites proximal to it, and may reflect both intrinsic effects through the covalent bond and the formation of a hydrogen bond between phosphate and the amide group [31,32], indicating that phosphorylation may affect the local structure in the vicinity of the phos- phorylated site. Identification of the hydrogen-bonding partners depends on detailed investigations into the pH depen- dence of their NMR parameters over the pH range 3–8. Obviously, the pH titration curve of the amide proton and the titration curve of 31 P of phosphory- lated serine residue have the almost identical pK values, which indicate hydrogen-bonding interactions between phosphate and the amide group (Fig. 2). An important result is that the titration parameters of the backbone amide proton of Ser356 remain virtually Fig. 1. Comparison of chemical shift differences of NH (white bar) and aH (black bar) between peptide R4 and phosphopeptide pR4 at pH 5.6 and 278 K. Changes in chemical shifts upon phosphory- lation (d phosphorylated –d nonphosphorylated ) are shown for each residue, positive values are downfield shifts and negative values are upfield shifts. Fig. 2. One-dimensional 31 P NMR spectra of pR4 with pH titration at 295K: (A) pH 3.0, (B) pH 3.9, (C) pH 4.9, (D) pH 5.6, (E) pH 6.6, (F) pH 7.5 (left). Changes with d 1 H NMR of amide protons in Ser356 and d 31 P NMR of the phosphate group during the pH titration of R4 and pR4 (right). Modulation of tau R4 peptide by phosphorylation J T. Du et al. 5014 FEBS Journal 274 (2007) 5012–5020 ª 2007 The Authors Journal compilation ª 2007 FEBS unchanged in nonphosphopeptide R4 compared with phosphopeptide pR4. In phosphopeptide, a phosphorylated serine acid side chain contains a single titratable group (phos- phate) with pK a1 and pK a2 . The pK a value is deter- mined from a fit of the phosphorus chemical shifts of the without ionic, monoionic and diionic forms of the phosphate group as a function of pH [33,34]. The phosphorus chemical shift change reflecting the equi- librium between without ionic and monoionic form (pK a1 ) is not observed in the pH range 3–8 [31,35]. The pK a values of phosphate group for the equilibrium between the monoionic and diionic form (pK a2 ) are obtained from the changes of the phosphorus chemical shift with pH titration (Fig. 2). Under acidic conditions, the phosphopeptide had one negative charge and an intraresidue hydrogen bond between the nearby amide proton and the phosphate group. As the pH increased, deprotonation began at the phosphate group, and the hydrogen bond began to surpass weakened electrostatic repulsion and led to the amide proton chemical shift downfield. Thus, the Ser356 amide proton chemical shift downfield in the phosphopeptide could be explained by the hydrogen bond between the nearby amide proton and the phos- phate group and deprotonation in the phosphate. In phosphorylated peptide pR4, a hydrogen bond between the nearby amide proton and the phosphate group appears to be the driving force behind the struc- tural changes that occur upon phosphorylation of Ser356. In addition, the NMR spectra in water sug- gested the presence, except for the major conformer, of one or more minor conformations for the R4 peptide, as evidenced by the appearance of additional reso- nances of lower intensity than those in the major con- former [21]. However, only one major conformation was observed in phosphopeptide pR4. CD spectra for R4 and pR4 are characterized by a strong negative apex at 198 nm (Fig. 3), which indicates a large amount of random coil structure [36]. No remarkable structural perturbation is suggested upon the phos- phorylation of Ser356. Effect of phosphorylation on assembly of the tau repeat An aggregating study was performed in NaCl ⁄ P i ,a buffer widely used to mimic physiological conditions [37,38]. Electron microscopy, turbidity and ThT fluo- rescence measurements confirm that both R4 and pR4 are capable of self-aggregation. The aggregation kinetics process was derived from the time dependence of turbidity at 405 nm. As shown in Fig. 4, both peptides showed little aggregation on day 1. However, the turbidity of both peptides increased sharply on the day 2, indicative of a nucle- ation step involved in the aggregation. Once the seed is formed, the filaments can form quickly. In addition, peptide R4 aggregated more quickly than phospho- peptide pR4 on days 2–4. During day 5, peptide R4 aggregated at almost the same speed as on day 4, whereas the aggregation speed of phosphopeptide pR4 increased. On day 6, the turbidity of both pep- tides had decreased somewhat, indicating that the aggregation had reached equilibrium. According to the kinetic turbidity curve, a different intrinsic rate of nucleation of aggregation is suggested. Filibration of R4 is considerably easier than that of pR4. In addition, the aggregation kinetics was also derived from the time dependence of the relative ThT fluores- Fig. 3. CD spectra of peptide R4 and phosphopeptide pR4. Fig. 4. Aggregation of peptide R4 and phosphopeptide pR4 as mon- itored by turbidity. Peptides were dissolved in NaCl ⁄ P i , pH 7.4 (137.0 m M NaCl, 3.0 mM KCl, 10.0 mM Na 2 HPO 4 and 2.0 mM KH 2 PO 4 , ionic strength 160.0 mM) to a final concentration of 1.0 mgÆmL )1 and incubated at room temperature. The assembly time course of peptide R4 and phosphopeptide pR4 is plotted ver- sus the incubation time according to the turbidity at 405 nm. J T. Du et al. Modulation of tau R4 peptide by phosphorylation FEBS Journal 274 (2007) 5012–5020 ª 2007 The Authors Journal compilation ª 2007 FEBS 5015 cence intensity at 485 nm in NaCl ⁄ P i (Fig. 5). As shown in Fig. 5, the rate of filament formation was much greater for R4 than for pR4, indicating non-iden- tical filament formation for the R4 and pR4 peptides. Electron microscopy was also used to evaluate the aggregation of peptide R4 and the effect of phosphory- lation modulation on the process. In contrast to the typical long filament of peptide R4, negatively stained images of polymerized phosphopeptide pR4 revealed some thinner filaments (Fig. 6). Electron microscopy confirmed that phosphorylation in Ser356 was able to modulate the aggregation form of R4 in vitro. Discussion Knowing what regions of the protein tau are involved in its aggregation into aberrant filaments and what molecular structure is induced by aggregation are criti- cal steps towards understanding the mechanisms involved in the pathological aggregation of tau. Tau protein purified from brain extracts or recombin- ant tau is able to aggregate in vitro at high protein concentrations [39–41]. However, it is difficult to study the mechanism of tau aggregation using the full-length tau molecule because some regions act as inhibitors of polymerization. Furthermore, even if full-length tau obtained by recombinant means was used, it does not mimic the phosphorylation state of tau molecules com- prising PHFs [42]. Therefore, despite the growing body of data suggesting that different domains of the pro- tein may have different secondary structures [43], we decided to approach the problem by studying these factors in small tau fragments. It has reported that fragments from the tubulin-binding motif of tau can assemble into filaments in vitro. At present, the contribution of the R4 repeat to PHF formation remains to be elucidated, even though the roles of the R2 and R3 repeats in the aggregation of tau have been reported [22]. Moreover, there is no firm con- clusion concerning the effect of phosphorylation on aggregation. In this study, we have shown that both peptide R4 and phosphopeptide pR4 are capable of self-aggregation without the need to add aggregation inducer in NaCl ⁄ P i , according to the results of electron microscopy, ThT fluorescence and turbidity experi- ments. This leads to the suggestion that the R4 repeat might also play an essential role in PHF formation in vivo. The ability of the R4 repeat to self-aggregate implies that R4 repeats in the microtubule-binding domain might recognize each other and facilitate aggre- gation of the tau protein. To better understand the mechanism of PHF for- mation, the effects of phosphorylation on the confor- mation and aggregation of the R4 repeat were studied. Introduction of the phosphate ion, which predominantly carries a double negative charge at neutral pH, affects the electrostatic potential and quite often the conformation of the modified protein. Even in the absence of rearrangement, the change in the electric field and steric hindrance from a phosphate group can have biologically significant Fig. 6. Electron microscopy images of in vitro filaments of peptide R4 (left) and phosphopeptide pR4 (right). The black bar in the figure represents 100 nm. Fig. 5. Time profiles of peptide R4 and phosphopeptide pR4 aggre- gations in NaCl ⁄ P i as monitored by relative ThT fluorescence inten- sity at 485 nm. Modulation of tau R4 peptide by phosphorylation J T. Du et al. 5016 FEBS Journal 274 (2007) 5012–5020 ª 2007 The Authors Journal compilation ª 2007 FEBS consequences, e.g. promoting or opposing protein– protein interactions. Phosphorylated side chains typi- cally carry a )2 charge at physiological pH, although the pK a of the phosphate group is  6, and the )1 species may be present in certain proteins or at low pH. Phosphorylation is a key cause of modification in cellular regulation. There is increasing evidence that phosphorylation may influence filament forma- tion in peptides and proteins [28,29]. In this study, phosphorylation at Ser356 exhibited a modulated effect on aggregation compared with peptide R4. This modulated effect of phosphorylation on aggregation from the tubulin-binding motif might offer some clues on its role in the progression of AD. The modulation of phosphorylation on aggregation might be essential for the aggregation of tau protein in vivo. The peptide concentration used in this study is much higher than in vivo, so the process of assembly is very clear in our experiments, that is, the time needed for aggregation is much shorter than in vivo. It is likely that phos- phorylation exerts its toxic effects in AD via different aggregation behavior of the tau protein. The different aggregation behavior of peptide R4 and phosphopeptide pR4 might be explained by con- sidering the different conformations of R4 and pR4. It has been reported that phosphorylation can modulate the structure of the first and third tau microtubule- binding repeat, which in turn results in a change in aggregation behavior [42,44]. For R4 and pR4, a local conformational difference was deduced from the pro- ton chemical shift deviation of NH and aH. However, there was no remarkable structural perturbation from the CD spectra. Furthermore, our study has confirmed that a hydrogen bond is formed between the phosphate and the amide proton of the phosphorylated serine res- idue in phosphopeptide pR4 [21]. The hydrogen bond is supposed to be the driving force behind the struc- tural changes that occur upon phosphorylation. So how does phosphorylation alter the aggregation behav- ior of peptide R4? A possible explanation is that phos- phorylation might affect the kinetics of conversion of the native structure to a filament-like structure [45]. In other words, phosphorylation might act through the hydrogen bond to alter the structural proclivity among different conformational states, which results in differ- ent aggregation behavior. In conclusion, it is shown that the R4 repeat is capa- ble of self-aggregation and phosphorylation at Ser356 can modulate the aggregation in the process of assem- bly, implying that R4 repeat might also play an impor- tant role in PHFs formation and phosphorylation at Ser356 might serve as an aggregation modulation in the progression of AD. Study such as this may be valuable in future research undertaken to clarify the pathophysiology of AD. Experimental procedures Peptide synthesis Peptides were synthesized on Fmoc-Wang resin using the standard Fmoc ⁄ tBu chemistry and HBTU ⁄ HOBt protocol [46]. For phosphopeptide, phosphoserine was incorporated as Fmoc-Ser(PO 3 HBzl)-OH [47]. Peptides and all protecting groups were cleaved from the resin with trifluoroacetic acid containing phenol (5%), thioanisole (5%), ethanedithiol (2.5%) and water (5%) for 120 min [48]. Crude peptides were purified by reverse-phase HPLC using an ODS-UG-5 column (Develosil) with a linear gradient of 20–50% aceto- nitrile containing 0.06% trifluoroacetic acid as an ionpairing reagent. The integrity of the peptide and phosphopeptide was verified by ESI-MS and NMR spectroscopy. The synthetic peptides are listed in Table 1. CD The peptides (1.0 mgÆmL )1 ) were dissolved in phosphate buffer, pH 7.6 (10.0 mm Na 2 HPO 4 ). CD spectra were recorded on a Jasco model J-715 spectropolarimeter (Jasco, Tokyo, Japan) at 298 K under a constant flow of nitrogen gas. Typically, a quartz cell with a 0.1 cm path length was used for spectra recorded between 190 and 250 nm with a 1-nm scan interval. CD intensities reported in the figure are expressed in mdeg. NMR spectroscopy Peptide samples for NMR measurements were dissolved in H 2 O ⁄ D 2 O9:1(v⁄ v) in 10.0 mm phosphate or sodium d 4 - acetic acid buffer. The pH value was adjusted by adding HCl or NaOH. Sodium d 4 -2,2-dimethyl-2-silapentonate in a capillary tube was used as the external standard for 1 H NMR chemical shifts. Standard NOESY [49] and TOC- SY [50] experiments were collected on a Varian Inova-600 spectrometer (Palo Alto, CA, USA) or a Jeol ECA-600 spectrometer (Tokyo, Japan). The 31 P NMR spectra were acquired on a Bruker ACP200 spectrometer with 85% phosphoric acid as the external reference. Two-dimensional NMR data were processed using the nmrpipe ⁄ nmrdraw program [51]. A sinesquared window function shifted by p ⁄ 4 ) p ⁄ 2 was applied in both dimensions, with zero filling in f1–2K points. Quadrature detection in f1 was achieved using time proportional phase incrementation [52]. H 2 O res- onance was suppressed either by presaturation of the sol- vent peak during the relaxation delay (and the mixing time in the NOESY spectra) or by using a pulsed-field gradient technique with a WATER-GATE sequence [53,54]. In J T. Du et al. Modulation of tau R4 peptide by phosphorylation FEBS Journal 274 (2007) 5012–5020 ª 2007 The Authors Journal compilation ª 2007 FEBS 5017 general, spectra were collected with 2K points in f2 and 512 in f1. Identification of hydrogen bond using pH titration experiments The protocol to identify hydrogen-bonding interactions between phosphate and the amide group was based on changing the pH from acidic to basic [33,34]. When a cer- tain amide proton was involved in such a hydrogen bond and downfield chemical shifts were observed during the pH variation, which indicated the hydrogen bond between the amide proton and phosphate. pH values were measured with a microcombination pH ⁄ sodium electrode (Orion Research, Inc., Beverly, MA) attached to an Orion 520A pH meter. Calibration of the pH meter was carried out at room temperature using pH 4.00 ± 0.01, pH 7.01 ± 0.01, and pH 10.00 ± 0.01 calibration buffers. Monitoring the aggregation of R4 and pR4 using turbidity Peptides (1.0 mgÆmL )1 ) were dissolved in NaCl ⁄ P i , pH 7.4 (137.0 mm NaCl, 3.0 mm KCl, 10.0 mm Na 2 HPO 4 , and 2.0 mm KH 2 PO 4 , ionic strength  160.0 mm). Identical methods were used to prepare peptide samples utilized for electron microscopy and ThT fluorescence experiments. To study aggregation, peptides (1.0 mgÆmL )1 ) were incubated at room temperature in a nonbinding surface 96-well plate. The aggregation was monitored each day at the same time via turbidity measurements at 405 nm on Wellscan MK3 instrument (Labsystems Dragon Co., MA, USA). Monitoring of aggregation of R4 and pR4 using ThT fluorescence Peptides (1.0 mgÆmL )1 ) were dissolved in NaCl ⁄ P i and incubated at room temperature. During the incubation, 20.0 lL aliquots of the reaction solutions were added to sodium phosphate buffer (700.0 lL) containing ThT (10.0 lm). Fluorescence spectra were collected using a Hit- achi F-4500 fluorescence spectrophotometer (Tokyo, Japan). An excitation frequency of 440 nm was used, and data were collected over the range of 450–600 nm. Samples were placed in a four-sided quartz fluorescence cuvette (Mu ¨ llheim, Germany), and data were recorded at room temperature. The excitation slit width was set at 5 nm and the emission slit width was set at 5 nm. The background fluorescence of the sample was subtracted when necessary. Transmission electron microscopy Filaments were viewed by electron microscopy. Negative staining of the sample was performed on formvar- and carbon-coated 300-mesh copper grids. Samples were loaded on the grid and left for 2 min for absorption and then stained with 1% tungstophosphoric acid for another 2 min. After drying in a desiccator overnight, the samples were viewed on a JEOL-1200EX electron microscope at 100 kV. 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Abbreviations AD, Alzheimer’s