Biochemistry 2005, 44, 6003-6014
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Aβ40-Lactam(D23/K28) Models a Conformation Highly Favorable for Nucleation of Amyloid† Kimberly L. Sciarretta,‡ David J. Gordon,§ Aneta T. Petkova,| Robert Tycko,| and Stephen C. Meredith*,§,⊥ Departments of Molecular Genetics and Cell Biology, Biochemistry and Molecular Biology, and Pathology, The UniVersity of Chicago, Chicago, Illinois 60637, and Laboratory of Chemical Physics, National Institute of Diabetes and DigestiVe and Kidney Diseases, National Institutes of Health, Bethesda, Maryland 20892-0520 ReceiVed NoVember 30, 2004; ReVised Manuscript ReceiVed February 10, 2005
ABSTRACT: Recent solid-state NMR data (1) demonstrate that Aβ1-40 adopts a conformation in amyloid fibrils with two in-register, parallel β-sheets, connected by a bend structure encompassing residues D23VGSNKG29, with a close contact between the side chains of Asp23 and Lys28. We hypothesized that forming this bend structure might be rate-limiting in fibril formation, as indicated by the lag period typically observed in the kinetics of Aβ1-40 fibrillogenesis. We synthesized Aβ1-40-Lactam(D23/K28), a congener Aβ1-40 peptide that contains a lactam bridge between the side chains of Asp23 and Lys28. Aβ1-40-Lactam(D23/K28) forms fibrils similar to those formed by Aβ1-40. The kinetics of fibrillogenesis, however, occur without the typical lag period, and at a rate ≈1000-fold greater than is seen with Aβ1-40 fibrillogenesis. The strong tendency toward self-association is also shown by size exclusion chromatography in which Aβ1-40-Lactam(D23/K28) forms oligomers even at concentrations of ≈1-5 µM. Under the same conditions, Aβ1-40 shows no detectable oligomers by size exclusion chromatography. Our data suggest that Aβ1-40Lactam(D23/K28) could bypass an unfavorable folding step in fibrillogenesis, because the lactam linkage “preforms” a bendlike structure in the peptide. Consistent with this view Aβ1-40 growth is efficiently nucleated by Aβ1-40-Lactam(D23/K28) fibril seeds.
β-Amyloid (Aβ)1 peptides are derived from the proteolytic cleavage of the β-amyloid precursor protein, forming Aβ1-40, Aβ1-42, and other less abundant products (2, 3). Aβ peptides, which constitute the most abundant protein component of † We thank the NIH Molecular Cell Biology Training Grant (NIH/ NIGMS T32 GM07183, KLS), American Federation of Aging Research (Glenn/AFAR 2004, KLS) and the NIH Medical Scientist Training Grant (5 T32 GM07281, DJG) and the Alzheimer’s Association (IIRG 98-1344, SCM) and NIH (RO1 NS042852, SCM) for support of this work. * Corresponding author. Mailing address: Department of Pathology, The University of Chicago, 5841 S. Maryland Ave., Chicago, IL 60637. Tel: 773-702-1267. Fax: 773-834-5251. E-mail: scmeredi@ uchicago.edu. ‡ Department of Molecular Genetics and Cell Biology, The University of Chicago. § Department of Biochemistry and Molecular Biology, The University of Chicago. | National Institutes of Health. ⊥ Department of Pathology, The University of Chicago. 1 Abbreviations: 1D, one-dimensional; Aβ, β-amyloid peptide; BOP, benzotriazolyl-N-oxytris-(dimethyl-amino)phosphonium hexafluorophosphate; DIEA, diisopropylethylamine; DMS, dimethyl sulfide; DMSO, dimethyl sulfoxide; EDT, ethanedithiol; ESI, electrospray ionization; FMOC, 9-fluorenylmethoxycarbonyl; fpRFDR-CT, finitepulse radio frequency driven recoupling; HBTU, 2-(1H-benzotriazol1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate; HFIP, hexafluoroisopropanol; HOBT, N-hydroxybenzotriazole; HPLC, high performance liquid chromatography; MAS, magic-angle spinning; MALDITOF, matrix-assisted laser desorption ionization time of flight; NHSoFMOC, 9-fluorenylmethyl succinimidyl carbonate; NMP, N-methylpyrrolidinone; PBS, phosphate buffered saline; REDOR, rotational echo double resonance; RP-HPLC, reverse-phase high performance liquid chromatography; tBOC, tert-butoxycarbonyl; TFA, trifluoroacetic acid.
neuritic plaques in Alzheimer’s disease, assemble into fibrils with high β-sheet content. In this respect, Aβ fibrils resemble those formed by other proteins involved in other neurodegenerative diseases, including Huntington, Parkinson, and prion diseases. Fibril-forming proteins lack sequence similarity, but the fibrils are believed to share some structural characteristics derived from their β-sheet content (4), such as protease resistance and the ability to bind thioflavin (5, 6) and Congo Red dyes (7-9). The kinetics of Aβ1-40 fibrillogenesis show a long lag phase, in which no fibrils are formed, followed by rapid polymerization of the peptide into fibrils. The lag phase has been attributed to a rate-limiting step that consists of highorder oligomerization and/or conformational changes (10, 11). Because of the high propensity of Aβ peptides to aggregate into insoluble fibrils and the temporal instability of intermediates in the pathway to fibril formation, traditional biophysical techniques are difficult to apply, and consequently little is know of the structure of these intermediates. A micelle-like oligomer has been proposed as an intermediate in Aβ fibrillogenesis (12, 13). Small oligomers have been studied in permeabilization of lipid bilayers suggesting a mechanism of pathogenesis (14). Small angle neutron scattering has demonstrated micelle-like intermediates in β-amyloid protein fibril assemblies; these have been shown to contain 30-50 Å monomers and have elongated geometries (15). In other studies, Aβ fibrillogenesis was shown to involve conformational changes leading to the formation of extended β-sheets and proceeding through an oligomeric R-helix-containing intermediate (16).
10.1021/bi0474867 CCC: $30.25 © 2005 American Chemical Society Published on Web 03/30/2005
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FIGURE 1: (Modified from Petkova et al. (1).) Structural model for Aβ1-40 fibril derived from energy minimization using constraints based on solid-state NMR data (1). (A) Peptide backbone conformation in the fibrils, showing a disordered N-terminal segment (blue), two β-strand segments (residues 10-22 and 30-40), and the bend segment (residues 23-29) containing oppositely charged Asp23 and Lys28. (B) Cross-sectional view of a piece of the Aβ1-40 protofilament, in which peptide molecules assemble into a fourlayered parallel β-sheet structure, stabilized by hydrophobic contacts within and between the two molecular layers.
We have taken a model peptide approach to study critical conformational changes in the fibrillogenesis pathway. Solidstate NMR data (17-20) have demonstrated that the peptides in fibrils formed by Aβ10-35 and Aβ1-40 are aligned in parallel, in-register, β-sheets. Earlier studies used dipolar recoupling NMR methods to measure distances in these peptides between labels (peptides singly or doubly labeled with 13C or 15N), mainly in the backbone. Peptides containing single 13C-carbonyl labels had intermolecular 13C-13C distances of approximately 5 Å at many positions in the peptide from Tyr10 to Val40. Results such as these were most consistent with parallel alignment of β-sheets. Chemical shift data also supported the conclusion of β-sheet structure for most residues in this region. More recently, a detailed structural model for fibrillar Aβ1-40 was proposed from continued data accumulation and constrained energy minimization (1). This model is consistent with data from NMR studies including measurements of intermolecular distances using fpRFDR-CT (19), DRAWS (17), MQ 13C NMR spectra (20), torsion angle constraints (21), and 13C and 15N chemical shift and line width measurements from 2D spectroscopy (1), as well as fibril mass per length from electron microscopy (22) and X-ray diffraction data (23). According to this model (shown in Figure 1), residues 10-22 and 30-40 form two β-strands in Aβ1-40 fibrils, which are connected by a bend structure encompassing residues D23VGSNKG29. This bend differs from a β-turn. Whereas a β-turn, defined by a set of backbone torsional angles, contains a hydrogen bond between backbone atoms, the bend structure in Aβ fibrils occurs in the “sidechain dimension” because it is defined and stabilized by sidechain interactions, both hydrophobic interactions and a salt bridge (between Asp23 and Lys28 in Figure 1), between the two separate, parallel β-sheets. Hence, a single molecular layer in this model is a double β-sheet. The first 10 residues of Aβ1-40 are structurally disordered in the fibril. According to this model, the protofilament could be constructed from two molecular layers, in which residues 30-40 create a hydrophobic surface for intermolecular interactions. Other investigators have also presented data in support of a non-
Sciarretta et al. β-sheet structure in the central portion of Aβ peptides. Solution-state NMR suggested a turn at positions 21-24 (24) or between 22-25, 24-27, 27-30, and 30-33 (25) of Aβ10-35, or 20-26 of Aβ1-40 (26) in soluble, monomeric form, while molecular modeling and molecular dynamic simulations also suggest a bend or turn in this region of Aβ10-35 (27). In addition, proteolysis experiments and hydrogen/deuterium exchange of Aβ1-40 fibrils support the model of an unstructured, solvent-exposed N-terminus, and a structured core in the remainder of the peptide in fibrils (28, 29). Proline mutagenesis (30) supported a model in which residues 22, 23, 29, and 30 appear to be involved in turn region. More recently, To¨ro¨k et al. (31) used electron paramagnetic resonance spectroscopy to confirm an inregister Aβ-sheet structure for Aβ1-40 fibrils, and demonstrated that the region around residues 23-26 of Aβ1-40 were more dynamic than surrounding β-sheet regions, which was compatible with a bend or turn in this region. In this paper, we first report solid-state NMR data for salt bridge formation from the side-chain interactions of residues Asp23 and Lys28. Thereafter, we test the hypothesis that a rate-limiting step in fibrillogenesis is the conformational change similar to the “bend” seen in the fibril. To test this hypothesis, we have designed a congener Aβ1-40 peptide that contains a lactam bridge between the side chains of Asp23 and Lys28, and thus is intended to model the structure proposed by Petkova et al. (1). If the congener peptide models a structure similar to that in the Aβ fibrils, we would predict that it would form fibrils resembling the un-crosslinked Aβ1-40 peptide, but at much greater rates. We show that this congener does indeed form fibrils similar to that of Aβ1-40 but at ≈1000-fold greater rate. We also demonstrate that fibril formation by this lactam-containing peptide occurs without a lag period, suggesting that fibrillogenesis by the lactam-containing congener of Aβ1-40 bypasses the ratelimiting step in fibrillogenesis of un-cross-linked Aβ1-40. EXPERIMENTAL PROCEDURES Peptide Synthesis and Purification. Aβ1-40 (NH2-DAEFRHDSGY10EVHHQKLVFF20AEDVGSNKGA30IIGLMVGGVV40-COOH) was synthesized using modified 9-fluorenylmethoxycarbonyl (FMOC) and HBTU/HOBT (Fastmoc) chemistry on an Applied Biosystems (Foster City, CA) model 433A instrument. Peptides were cleaved from the resin using 9 mL of TFA plus 0.5 mL of thioanisole, 0.3 mL of EDT, and 0.2 mL of anisole for 1.5 h at 22 °C. Peptides were purified by RP-HPLC on a preparative C18 (Zorbax) column at 60 °C. Peptide purity was greater than 98% by analytical HPLC. The molecular mass of the peptide was verified by ESI and MALDI-TOF mass spectrometry. A congener of Aβ1-40 was synthesized with an amide cross-link between the side chains of Asp23 and Lys28 (Aβ1-40-Lactam(D23/K28)) using standard tBOC chemistry. The procedure for forming the lactam cross-link is based on the methods described in detail elsewhere (32). Briefly, the peptide chain was elongated using tBoc chemistry; R-BocAsp(β-OFm)-OH (Bachem) and R-Boc-Lys(-Fmoc) (Bachem) were used for residues to be cross-linked. After Asp23 had been incorporated into the chain (R-BOC group remaining on Asp23), the Asp23 and Lys28 side chains were deprotected with piperidine (20%, v/v in NMP, 23 min,
Aβ40-Lactam(D23/K28) Models a Nucleating Conformation 22 °C) and coupled using benzotriazolyl-N-oxytris-(dimethylamino)phosphonium hexafluorophosphate (BOP) reagent (33), with the addition of DIEA and NMP for 4 h to form an amide bond between the β-carbon of Asp23 and the -nitrogen of Lys28. Coupling continued for 2 h, after which the reaction vessel was drained, additional BOP, NMP, and DIEA were added, and the reaction was allowed to proceed for another 2 h. Formation of the cross-link was later confirmed by ESI and MALDI-TOF mass spectrometry. After formation of the lactam cross-link, the peptide-resin was replaced in the synthesizer, and the remainder of the peptide chain was elongated using standard tBOC chemistry. For solid-state NMR experiments, 1-13C-valine was incorporated as Val18 of this peptide. 1-13C-Val (g97% isotopic purity) was purchased from Cambridge Isotopes (Andover, MA) and was protected with the FMOC group by Midwest Biotech. Peptides made by tBOC chemistry were cleaved from the resin using anhydrous HF in an Immuno-Dynamics (La Jolla, CA) HF apparatus, using 10 mL of HF also containing 1 mL of p-cresol and 1 mL of DMS per 1 g of resin for 1 h at -3 to -5 °C. A 0.5 mmol scale reaction gave approximately ≈200 mg of crude peptide, of which only ≈5-8 mg of purified product could be obtained. The low yield was attributable to the high propensity of the peptide to aggregate, which made it difficult to separate the peptide from impurities. Nevertheless, a procedure was found to isolate pure peptide, as follows. Peptide solubilized by being stirred into 40:60 ) acetonitrile:water (both containing 0.1% TFA, v/v) at ≈60 °C was chromatographed using a Zorbax C-18 preparative column maintained at 60 °C and eluted from the column using a 50 min gradient from 25 to 65% (v/v) acetonitrile containing 0.1% (v/v) TFA. Purity was assessed by analytical RP-HPLC using a Vydac C-18 column. Mass of the product was verified using MALDITOF and ESI mass spectrometry. Kinetics of Fibrillogenesis. The purified Aβ peptides were stored as follows. Peptide was dissolved at ≈50 °C into 30: 70 acetonitrile:water containing 0.1% TFA (v/v) and lyophilized as aliquots of 0.5 mg in siliconized 1.5 mL tubes, and then it was stored at -20 °C. For fibrillogenesis assays, peptide concentration in buffer was assessed from absorbance at 274.6 nm, using the extinction coefficient for tyrosine of 1420 M-1 cm-1. Fibrillogenesis was followed by thioflavin fluorescence at 37 °C. At various time points, 10 µL aliquots were taken, diluted into 1 mL of 10 µM Thioflavin T solution, in 10 mM sodium phosphate buffer, pH 7.40. The sample was pipetted vigorously, and fluorescence was monitored. Measurements (λEX ) 446 nm, λEM ) 490 nm) were made on a Hitachi F2000 fluorescence spectrophotometer and, after a 3 s delay, were averaged for 10 s with a bandwidth of 10 nm and a photomultiplier voltage of 700. To compare the fibrillogenesis kinetics of Aβ1-40-Lactam(D23/K28) with those of Aβ1-40 we first compared a number of fibrillogenesis conditions for Aβ1-40 to determine which gave the most reproducible kinetics, and most closely approximated “seed-free” conditions. A number of different techniques have been proposed for pretreating Aβ1-40 peptides to ensure reproducibility of kinetics, where monomer is predominantly or exclusively present at the initiation of the reaction. We assessed multiple conditions, using various combinations of the following: (1) solvents to induce disaggregation (neat HFIP, neat DMSO, neat TFA, 1 mM
Biochemistry, Vol. 44, No. 16, 2005 6005 NaOH); (2) sonication (varying time from 0 to 4 h); (3) removal of above solvents (by complete drying with N2, or dilution to
