Laboratorio de Biofísica Molecular, Facultad de Ciencias Biológicas, Universidad de Concepción, Concepción and 1 Departamento de Biología Celular y Molecular, Facultad de Ciencias Biológicas, Pontificia Universidad Católica de Chile, PO Box 114-D, Alameda 340, Santiago, Chile
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Abstract |
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Keywords: Alzheimer's disease/amyloid/modeling/triosephosphate isomerase
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Introduction |
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In vitro studies with Aß have shown that it presents a high aggregation capacity dependent on pH (Barrow and Zagorski, 1991; Esler et al., 1996
), concentration, ionic strength (Hilbich et al., 1991
; Lee et al., 1995
) and the presence of other proteins (Inestrosa et al., 1996
; Harper and Lansbury, 1997
). The aggregation process starts with a nucleation step followed by a growth phase, which is dependent on the composition of the carboxyl end of Aß (Jarrett et al., 1993
). X-ray studies of Aß fibrils have shown an anti-parallel ß-pleated sheet structure (Kirschner et al., 1986
, 1987
; Gorevic et al., 1987
), which is formed by four-stranded ß-pleated sheets, with repetitions of 10.6 Å between stacked sheets and 4.8 Å distance along the fiber axis. The amino acid sequence of Aß was originally determined by Glenner and Wong (1984) and the secondary structure was predicted by the ChouFasman algorithm as follows: 14,
-helix; 58, turn; 913,
-helix or ß-strand; 1418,
-helix; 1922, ß-strand; 2327, turn; 2840 ß-strand (; Gorevic et al., 1987;
Kirschner et al., 1987
). This prediction points to a helical conformation for the N-terminus and a ß-strand conformation for the C-terminus with hydrophobic character, although it has yet to be supported experimentally. During the Aß aggregation process, the
-helical segment of the N-terminus is predicted to convert to a ß-strand structure (Barrow and Zagorski, 1991
; Zagorski and Barrow, 1992
; Sorimachi and Craik, 1994
; Soto et al., 1995
).
The structure of fragment 128 of Aß has been solved by nuclear magnetic resonance (NMR) spectroscopy (Zagorski and Barrow, 1992; Sorimachi and Craik, 1994
; Talafous et al., 1994
). Zagorski and Barrow (1992) observed that various regions of Aß128 (i.e. residues 227 or residues 27 and 1027) can adopt an
-helical conformation. Later, Sorimachi and Craik (1994) reported that, although the fragment Aß128 can exist as a mixture of conformations in solution, one of such population of structures possesses a flexible N-terminal region and a well defined C-terminal region, which includes an
-helical segment and a terminal turn-like structure. Finally, Talafous et al. (1994), working in a membrane-like medium (resembling the structure under physiological conditions), showed that the solution structure of the Aß128 fragment is almost entirely
-helical (consisting of two right-handed
-helices, residues 211 and 1327, which are connected by a bend centered at Val12).
Regarding the conformation of the Aß140 C-terminal region, the synthesis of analog peptides containing a disulfide bridge, followed by infrared (IR) and circular dichroism (CD) spectroscopy, have demonstrated that fragments 2942, 2243 and 143 adopt the conformation of two antiparallel ß-sheets with a central ß-turn at residues 2629 (Hilbich et al., 1991; Hughes et al., 1996
). Hydrophobic interactions at Phe19 and Phe20 seem to be important for the aggregation process and this effect is increased by the presence of His13 and His14 at neutral pH (Hughes et al., 1996
).
Efforts to obtain the complete three-dimensional structure of Aß, under physiological conditions, so far have been unsuccessful. Sticht et al. (1995) reported that the Aß140 peptide NMR structure, at pH >2.8, presented two helical regions (Gln15 to Asp23 and Ile31 to Met35) that showed no interactions between them. On the other hand, two models for the structure of the Aß peptide have been proposed. Whereas Soto et al. (1994) considered hydrophobicity profiles to identify secondary structure predictors and computational analysis (energy minimization) to generate a model for soluble Aß1-42, Iversen et al. (1995) proposed a possible arrangement of Aß1-42 monomers to form an anti-parallel ß-structure. Here, we have modeled the complete structure of the Aß peptide based on the 128 segment of Aß solved by NMR and by the homology of the peptide to a segment of triosephosphate isomerase (TIM). We have also shown that this segment of TIM is able to form amyloid.
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Materials and methods |
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Peptide fragments derived from the human wild-type Aß140 peptide were obtained from Sigma Chemical (St. Louis, MO) and from Chiron (Emeryville, CA). Fragment 186218 derived from Escherichia coli TIM was synthesized by using solid-phase techniques at the Technical Institute, Braunschweig, Germany. All peptides were purified by reversed-phase high-performance liquid chromatography and their purity was evaluated by amino acid sequence analysis.
Computational methods
An extensive sequence homology search for Aß peptide was done using the Smith and Waterman algorithm (Smith and Waterman, 1981; Shomer et al., 1996
) implemented in MPsrch (Blitz server http://www.ebi.ac.uk/searches/blitz_input.html) against the Swiss Protein database. The selected proteins were checked in the Protein Data Bank (Bernstein et al., 1977
) for three-dimensional structures. Multiple sequence alignments for these proteins were performed with the Pattern Induced Multi-sequences Alignment (PIMA) program, using a covering pattern construction algorithm (Smith and Temple, 1992
; Defay and Cohen, 1995
). This information yielded reference structures useful for assignment of atomic coordinates to 34 residues at the C-terminal region of the Aß sequence. The structure of the Aß128 fragment (PDB code: 1amb) solved by NMR (Talafous et al., 1994
) was used to assign coordinates to the N-terminus. The connecting loop, residues Ala22Gly30 of the Aß sequence, was chosen from the loop data bank of Homology (Biosym/MSI). Crystal structures of TIM from human (PDB code: 1hti) (Mande et al., 1994
), Trypanosoma brucei (4tim) (Noble et al., 1991
), chicken (1tpb) (Zhang et al., 1994
), Saccharomyces cereviase (1ypi) (Lolis et al., 1990
) and E.coli (1tre) (Kishan et al., 1994
) were used as references.
Refinement of side chain dihedral angles and other local optimizations were made to convergence (0.01 kcal/mol) using AMBER 4.1 (Weiner and Kollman, 1981, 1986
). Global optimizations were carried out first in vacuo and afterwards in aquo with Aß in a shell of water molecules located up to a maximum distance of 25.0 Å (SOL, IGROUP1 = CAP options of EDIT module) from the arbitrary center of the peptide at CG His13, leading to a system containing 1263 water molecules. PROCHECK v. 2.1.4 (Laskowski et al., 1993
) was employed to check the stereochemical quality of the optimized structures. All calculations and graphic visualizations were performed on an INDY machine using the Homology module and InsightII (Biosym/MSI).
Aggregation studies of TIM peptide: turbidity assay
Lyophilized aliquots of the TIM peptide were dissolved in dimethyl sulfoxide at 15 mg/ml (3.5 mM). Aliquots of peptide stock (70 nmol in ~20 µl of dimethyl sulfoxide) were added to phosphate-buffered saline, pH 7.3, in a final volume of 725 µl. Alternatively, aliquots of TIM peptide were resuspended in 0.1 M sodium acetate at pH 5.0. Incubations were carried out at room temperature and the solutions were stirred continuously (210 r.p.m.). At various times, aggregation was measured by turbidity at 405 nm versus buffer blank as described previously (Jarrett et al., 1993; Soto et al., 1995
; Inestrosa et al., 1996
).
Amyloid detection by Congo red (CR) measurements
The binding of CR to TIM aggregates was measured as described previously (Klunk et al., 1989) to quantify amyloid formation. Aliquots (40 µl) were added to 960 µl of a solution containing 25 µM CR, 100 mM phosphate (pH 7.4), 150 mM NaCl and incubated for 30 min. Absorbance was measured at 480 and 540 nm and CR binding was calculated as follows: CR (M) = (A540/25295) (A480/46306).
Electron microscopy of amyloid fibrils
The TIM peptide (1 mg/ml) was incubated in 0.1 M sodium acetate (pH 5.0) and the Aß peptide (1 mg/ml) in 0.1 M TrisHCl (pH 7.4), for 5 days at room temperature. The amyloid fibrils formed were then transferred on to Formvar carbon-coated 300-mesh nickel grids, negatively stained with 2% uranyl acetate for 1 min and examined under a JEOL 100-B electron microscope at 80 kV (Alvarez et al., 1997).
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Results |
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MPsrch used for homology search with Aß peptide yielded the results shown in Table I. A general low identity of 23.2% was obtained. Of the 27 non-amyloid proteins retrieved, only the sequence corresponding to a TIM enzyme from Culex tarralis with 22% local identity (Table I
, entry 23) was selected to provide atomic coordinates for most of the Aß sequence. The TIM fragments mainly pointed to a C-terminal homology with the Aß peptide. As the three-dimensional structure of TIM from C.tarralis has yet to be solved, we decided to search for related available structures. The family of TIM proteins contains several members, from different sources with known three-dimensional structures, namely 1hti, 4tim, 1tpb, 1ypi and 1tre.
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A multiple sequence alignment of the TIM family members is shown in Figure 1. The alignment shows three regions (I, II and III) homologous to the Aß peptide. The sequence of TIM from E.coli (1tre) contained two regions with the highest identity with Aß (20%); these regions (I and III) were selected to perform a new alignment assay with Aß (Figure 2
). The new local alignment increased the percentage identity of region I to 21%, but introduced two gaps into the Aß sequence, one of which is located in the loop zone of Aß. The alignment with region III did not introduce gaps into the Aß sequence and its C-terminal region showed a similar secondary structure. On the other hand, visual inspection of the three-dimensional structure of TIM in these regions showed that only regions II and III presented an
-helixloopß-strand structure, in agreement with previous CD and IR determinations. All these considerations induced us to choose site III of 1tre to model the structure of Aß.
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The N-terminal of region III in E.coli TIM (1tre) presented a high structural identity with residues 721 of the Aß NMR structure 1amb [0.664 Å root mean square deviation (r.m.s.d.)] (Figures 3 and 4) and the highest identity (40%) was obtained for the ß-strand segment Ala207Gly216. Sequence gaps arose only at a single loop between residues His199 and Ile206 of 1tre that would correspond to the ß-turn between residues Ala22 and Gly30 of the Aß peptide (Figure 4
). In this way, we used the atomic coordinates from 1amb for the first 21 residues of the
-helix N-terminal segment of Aß, and the ß-strand residues (Ala207Gly216) of 1tre for the C-terminal domain of the Aß model. The coordinates for the loop between residues Ala22 and Gly30 of Aß were obtained from the Loops Bank, which showed a segment of 0.463 Å r.m.s.d. The report of collision between atoms, obtained after coordinate assignment (Table II
), was resolved by optimization of the model.
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Energy refinements were made first on the colliding atoms by relaxing them. A global optimization moved the structure to a conformation of 1594.9 cal/mol and 0.098 Å r.m.s.d. A new step of energy minimization was developed with the system in a water shell (Jorgensen et al., 1983). The final conformation reached an energy of 17459.9 cal/mol and 0.095 Å r.m.s.d. against the conformation prior to global optimization. Further calculations of the optimization output in which dihedral angle contributions were neglected, showed a lower energy for the protein alone (1656 cal/mol) indicating stabilization due to the water environment. Optimization with aqueous solvent showed that water molecules mainly stabilized negatively charged residues. The low deviation values indicated that the final model remained very close to the reference conformations used for the N- and C-termini. The r.m.s.d.s for C
atoms were of about the same magnitude using either the optimization in vacuo (Figure 5
) or in aquo. The main deviations corresponded to the N- and C-terminal residues and to Gln15 and Leu34. PROCHECK (Laskowski et al., 1993
) reported only two non-glycine residues: Ala30 and Ile32 located in the disallowed region of the Ramachandran plot (Figure 6
).
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A number of studies with synthetic Aß peptides and Aß fragments in vitro have shown that this peptide aggregates and forms amyloid fibrils similar to those found in the brains of patients with Alzheimer's disease (Castaño et al., 1986; Kirschner et al., 1987
; Alvarez et al., 1997
). In view of our modeling studies with the Aß peptide, we asked whether or not part of the 1tre TIM site III was able to form amyloid. Figure 7A
shows turbidimetric measurements for the TIM aggregation process (Jarrett et al., 1993
), which clearly demonstrate that the TIM fragment is able to aggregate, although to a lesser extent than Aß. To confirm that amyloid was in fact formed, as indicated in Figure 7B
, Congo red, a dye known to bind amyloid, was used (Klunk et al., 1989
). Finally, negatively stained TIM fragments revealed that the aggregates were indeed composed of fibrils. No morphological differences were detected between the amyloid fibrils formed by Aß alone (Figure 8a
) and by the TIM site III fragment (Figure 8b and c
). In both cases, the amyloid fibrils showed the typical features: 710 nm thick unbranched fibrils, up to 23 mm in length (Castaño et al., 1986
; Kirschner et al., 1987
). The above results indicate that the 1tre TIM site III forms amyloid fibrils in much the same way as Aß140.
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Discussion |
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Energy optimization considering the water shell (17459.9 cal/mol) allowed the system to reach an additional stability compared with that determined in vacuo (1594.9 cal/mol). However, the difference in r.m.s.d. between the two systems was minimal (0.003 Å) indicating a high stabilizing effect on A due to the aqueous solvent. A more detailed energy analysis in which all dihedral angle contributions were neglected indicated that the strongest contribution was provided by the electrostatic interactions between charged residues and the water shell. In this system, the negatively charged residues made a stronger contribution than positively charged residues, whereas in contrast, optimization in vacuo revealed that the total energy was distributed uniformly along the chain of the Aß peptide. The highest deviation values corresponded to residues located at the N- and C-termini of Aß, a finding which is in accordance with the end-terminal flexibility generally observed in peptides. Residues Gln15 and Leu34 demonstrated a higher mobility than other residues, suggesting that these residues could play a role as hinges. The non-glycine residues Ala30 and Ile32 reported in the disallowed region of the Ramachandran plot (Figure 6
) correlated with the fact that these residues presented some of the lowest energy values (<31. 4 kcal/mol) detected for the same residues either in vacuo or in aquo. However this observation does not allow us to distinguish whether these residues are in an energy minimum as result of the complete peptide structure optimization or in a local minimum near to the start conformation in which the residues could be trapped.
Finally, the finding that part of site III of 1tre TIM was able to assemble into amyloid fibrils, as also occurs with Aß fragments (Gorevic et al., 1987; Alvarez et al., 1997
), provided strong biological evidence for our homology studies and gave further support to our choice of structural TIM domains for the construction of the Aß peptide model presented in this paper.
Conclusions
The model structure proposed here for the Aß peptide accounts for the biological characteristics of this molecule, including its aggregation properties. This work is the first step in our approach to studying the stability of the Aß peptide as monomers and dimers under physiological conditions, its interaction with other proteins which may play a role in the assembly and toxicity of amyloid plaques and the potential conformational changes involved in amyloid formation and the development of Alzheimer's disease at the molecular level.
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Acknowledgments |
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Notes |
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References |
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Received October 19, 1998; revised July 20, 1999; accepted August 2, 1999.