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Originally published In Press as doi:10.1074/jbc.M303189200 on June 9, 2003

J. Biol. Chem., Vol. 278, Issue 36, 34259-34267, September 5, 2003
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Folding and Stability of the Extracellular Domain of the Human Amyloid Precursor Protein*

Michelle G. Botelho {ddagger}, Matthias Gralle {ddagger}, Cristiano L. P. Oliveira § ¶, Iris Torriani § ¶ and Sérgio T. Ferreira {ddagger} ¶ ||

From the {ddagger}Department of Medical Biochemistry, Federal University of Rio de Janeiro, Rio de Janeiro, RJ 21944-590, Brazil, §Instituto de Física "Gleb Wataghin," Unicamp, Campinas, SP 13084-971, Brazil, and Laboratório Nacional de Luz Síncrotron, Campinas, SP 13084-9701, Brazil

Received for publication, March 27, 2003 , and in revised form, June 2, 2003.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
The {beta}-amyloid peptide (A{beta}), the major component of the senile plaques found in the brains of Alzheimer's disease patients, is derived from proteolytic processing of a transmembrane glycoprotein known as the amyloid precursor protein (APP). Human APP exists in various isoforms, of which the major ones contain 695, 751, and 770 amino acids. Proteolytic cleavage of APP by {alpha}- or {beta}-secretases releases the extracellular soluble fragments sAPP{alpha} or sAPP{beta}, respectively. Despite the fact that sAPP{alpha} plays important roles in both physiological and pathological processes in the brain, very little is known about its structure and stability. We have recently presented a structural model of sAPP{alpha}695 obtained from small-angle x-ray scattering measurements (Gralle, M., Botelho, M. M., Oliveira, C. L. P., Torriani, I., and Ferreira, S. T. (2002) Biophys. J. 83, 3513–3524). We now report studies on the folding and stabilities of sAPP{alpha}695 and sAPP{alpha}770. The combined use of intrinsic fluorescence, 4–4'-Dianilino-1,1'binaphthyl-5,5'-disulfonic acid (bis-ANS) fluorescence, circular dichroism, differential ultraviolet absorption, and small-angle x-ray scattering measurements of the equilibrium unfolding of sAPP{alpha}695 and sAPP{alpha}770 by GdnHCl and urea revealed multistep folding pathways for both sAPP{alpha} isoforms. Such stepwise folding processes may be related to the identification of distinct structural domains in the three-dimensional model of sAPP{alpha}. Furthermore, the relatively low stability of the native state of sAPP{alpha} suggests that conformational plasticity may play a role in allowing APP to interact with a number of distinct physiological ligands.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Alzheimer's disease is the most important cause of dementia worldwide. Cerebral deposits of the amyloid {beta} protein (A{beta})1 are the major pathological hallmark of Alzheimer's disease (for recent reviews, see Refs. 1–3). A{beta} is generated by proteolytic cleavage of the amyloid precursor protein (APP), a ubiquitous transmembrane protein that spans the membrane a single time (4) (see also Fig. 1) and exists in three main isoforms with 695, 751, or 770 amino acids (known as APP695, APP751, and APP770, respectively). The isoforms with 751 and 770 amino acids contain a Kunitz-type protease inhibitor (KPI) domain (5). The 695-amino acid isoform lacks the protease inhibitor domain and is predominant in neurons (6).



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  		FIG. 1.
Domain structure and cleavage of APP. APP, the entire transmembrane protein, numbered as in APP770. The signal peptide (residues 1–17) was omitted. The KPI domain (1AAP [PDB] .PDB) (33), which is present in APP770, is shown above its insertion site. HBD1 and HBD2, putative heparin-binding domains (HBD1 is equivalent to 1MWP [PDB] .PDB) (34). CuBD, copper-binding domain (35). ZnBD, putative zinc-binding domain. (DE)n, Asp- and Glu-rich region. RC, predicted random coil region. TM, transmembrane domain. A{beta}, amyloidogenic sequence (shaded in gray), which reaches partially into the transmembrane domain. Cleavage by {beta}-secretase at its N terminus and {gamma}-secretase at its C terminus gives rise to the A{beta} peptide. Cyt, cytoplasmic domain of APP. Cleavage by {alpha}-secretase releases the soluble sAPP{alpha} fragment (numbered as in sAPP{alpha}770), carrying, at its C terminus, part of the amyloidogenic sequence (gray). The membrane-bound C-terminal stub can be further cleaved by {gamma}-secretase (at the C terminus of the gray amyloidogenic sequence) into the p3 peptide (gray) and the cytoplasmic domain (Cyt). The diagram shows that sAPP{alpha}770 is equivalent to APP18-687 (670 amino acid residues), whereas sAPP{alpha}695 has 595 residues.

 

APP is proteolytically processed by a set of enzymes known as secretases. Cleavage of APP by {alpha}-secretase at a site located 13 amino acid residues upstream from its membrane insertion gives rise to a soluble secreted form of APP (sAPP{alpha}) and to a C-terminal fragment of 83 amino acids (Fig. 1) (7). Alternatively, cleavage by {beta}-secretase occurs at a site located 29 amino acids upstream from the membrane insertion point of APP, giving rise to secreted sAPP{beta} and to a C-terminal fragment with 99 amino acids that encompasses the whole A{beta} peptide sequence (8). Subsequent cleavage of this C-terminal fragment by {gamma}-secretase releases the A{beta} peptide.

In addition to its involvement in the production of A{beta} and, hence, in the pathogenesis of Alzheimer's disease, APP has been proposed to play several relevant physiological roles in cell proliferation (9), cell adhesion (10), and protection against excitotoxicity (11). Surprisingly, however, very little is known about the structure of APP or its folding and stability.

Using synchrotron radiation small angle x-ray scattering (SAXS) measurements, we have recently obtained the first structural model of the extracellular domain of human APP (12). Combined analysis of SAXS, CD, and size exclusion chromatography data revealed that sAPP{alpha}695 is monomeric in solution and exhibits an elongated shape. Analysis of CD data and secondary structure predictions based on the primary sequence of APP further indicated that sAPP{alpha} contains distinct folded domains and regions with little ordered structure. We now report studies of the equilibrium unfolding of sAPP{alpha}695 and sAPP{alpha}770 by guanidine hydrochloride (GdnHCl) and urea, monitored by a combination of CD, intrinsic and bis-ANS fluorescence, UV absorption, and SAXS measurements. Taken together, these results indicated a multistep folding pathway for sAPP{alpha} and revealed the existence of partially folded intermediate states in the unfolding by GdnHCl.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Materials—Recombinant human sAPP{alpha}695 and sAPP{alpha}770 were expressed in Pichia pastoris and purified as previously described (12). Dithiothreitol, guanidine hydrochloride, and urea were purchased from Sigma. Bis-ANS (Molecular Probes, Inc., Eugene, OR) was dissolved in methanol and then diluted to 1 mM in water for storage at 4 °C. NAP-25 columns were purchased from Amersham Biosciences.

Ultraviolet Absorption Measurements—sAPP{alpha} (7.17 µM) was diluted in 50 mM Tris-Cl, pH 7.4, in the presence of increasing concentrations of denaturants (urea or guanidine hydrochloride), as indicated under "Results." UV absorption spectra were measured at 25 °C on an Ultrospec 2000 spectrophotometer (Amersham Biosciences) using 1-cm path length semimicro quartz cells. Blank and protein samples were continuously alternated, and the subtracted spectra were accumulated (24 integrations) using the manufacturer's software. The scattering contribution was determined from absorption measurements between 314 and 324 nm and subtracted from the raw data to yield corrected sAPP{alpha} absorption spectra. Because the extinction coefficient at 270 nm is independent of experimental conditions (13), spectra were normalized for their absorption at 270 nm. Differential absorption spectra were generated by subtracting the spectra of sAPP{alpha} in the presence of denaturant from the spectrum of native sAPP{alpha}, and denaturation was monitored by the change at 286 nm, where the largest differences were seen.

Fluorescence Measurements—Fluorescence emission spectra were measured at 25 °C on Hitachi F-4500 (Tokyo, Japan) and ISS PC1 (Champaign, IL) spectrofluorometers. For intrinsic fluorescence measurements, the excitation was at 280 nm and emission was recorded from 300 to 420 nm, using 5-nm (F-4500) or 8-nm (ISS PC1) band passes for both excitation and emission. Bis-ANS fluorescence was measured with excitation at 365 nm and emission from 400 to 600 nm. All experiments were carried out in 50 mM Tris-Cl, pH 7.4, in the presence of increasing concentrations of denaturant, as indicated under "Results."

The fluorescence spectral center of mass (average emission wavelength, {lambda}av) was calculated according to the equation,

(Eq. 1)
where {lambda} is the emission wavelength, and I({lambda}) represents the fluorescence intensity at wavelength {lambda}.

Circular Dichroism—sAPP{alpha} isoforms were diluted to a concentration of 0.1 mg/ml in 50 mM Tris-Cl, pH 7.4, with increasing concentrations of denaturants (urea or guanidine hydrochloride), as indicated under "Results." Spectra were measured at 25 °C on a Jasco J-715 spectropolarimeter using a 0.1-cm path length quartz cell. Alternatively, 0.5 mg/ml solutions were measured using a 0.02-cm path length quartz cell on a Jasco J-810 instrument (at Laboratório Nacional de Luz Síncrotron). Unfolding was monitored by the loss of residual molar ellipticity at 222 nm.

Small Angle X-ray Scattering—SAXS experiments were performed at the D11A-SAXS beamline at the Laboratório Nacional de Luz Síncrotron in Campinas, Brazil (14). The monochromatic beam was tuned at 1.488 Å to minimize absorption by carbon atoms. The experimental setup included a temperature-controlled glass capillary and a Gabriel type two-dimensional position-sensitive detector. The buffer solution used was 50 mM Tris-Cl, pH 7.4, with or without denaturants, as indicated under "Results." The samples were kept at 10 °C during the measurements. More diluted protein samples were also measured to investigate possible concentration effects in the SAXS curves. Data acquisition was performed by taking several 600-s frames of each sample, which allowed control of any possible radiation damage. Several sample-detector distances enabled detection in the q range accessible within the given experimental conditions: 0.0328 Å–1 < q < 0.8538 Å–1 for sAPP{alpha}695 and 0.0241 Å–1 < q < 0.8609 Å–1 for sAPP{alpha}770. Data treatment was performed using the software package TRAT2D.2 Usual corrections for detector homogeneity, incident beam intensity, sample absorption, and blank subtraction were included in this routine. The output of this software provides the corrected intensities and error values. Data fitting was performed using the GNOM computer program (15).


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
Intrinsic Fluorescence Measurements—Fig. 2A shows intrinsic fluorescence emission spectra of native sAPP{alpha}695 (solid line), in the presence of 6.8 M GdnHCl (dashed line) or 8 M urea (dotted line). Native sAPP{alpha}695 exhibited maximum emission at ~337 nm, and significant red shifts were observed in the presence of both GdnHCl and urea, indicating increased exposure of the tryptophan residues of APP to the aqueous medium. The fluorescence emission spectra of native sAPP{alpha}770 and sAPP{alpha}770 in the presence of denaturants exhibited essentially the same behavior as observed for sAPP{alpha}695 (data not shown). The equilibrium unfolding of sAPP{alpha} by GdnHCl was monitored by the red shift in the fluorescence spectral center of mass ({lambda}av) in the presence of increasing concentrations of denaturant (Fig. 2B). Low concentrations of GdnHCl (<0.5 M) induced a small red shift (~1–2 nm) of the emission, and an initial plateau was observed at ~1 M GdnHCl. Higher GdnHCl concentrations (from ~1.5 to 2.5 M) induced a further fluorescence red shift (~3–4 nm), and a second plateau was observed at ~3 M GdnHCl. A third transition occurred at GdnHCl concentrations higher than 5 M, but a final plateau corresponding to fully unfolded sAPP was not achieved up to 7 M GdnHCl. Complete unfolding of both sAPP isoforms was obtained upon incubation for 30 min at 100 °C in the presence of 6 M GdnHCl and 1 mM dithiothreitol (Fig. 2B). It is important to note that full refolding of sAPP was obtained by running the protein through a desalting NAP-25 column in order to remove the denaturant (open symbols in Fig. 2B), indicating the complete reversibility of the (un)folding transition of both sAPP isoforms.



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  		FIG. 2.
Unfolding of sAPP{alpha}695 and sAPP{alpha}770 by guanidine or urea monitored by intrinsic fluorescence. sAPP{alpha} (0.25 µM) was diluted in 50 mM Tris-Cl (pH 7.4). A, fluorescence emission spectra of native sAPP{alpha}695 (solid line) in the presence of 6 M GdnHCl (dashed line) and in the presence of 8 M urea (dotted line). Spectra are normalized for maximum fluorescence emission. B, fluorescence spectral center of mass ({lambda}av) of sAPP{alpha}695 (filled circles) and sAPP{alpha}770 (filled triangles) as a function of increasing concentrations of GdnHCl. Complete unfolding was obtained by incubating the two isoforms in the presence of 6 M guanidine and 1 mM dithiothreitol at 100 °C for 30 min (cross-hair, sAPP{alpha}695; diamond, sAPP{alpha}770). Refolded samples were obtained by removal of GdnHCl using a desalting column, as described under "Results" (open circle, sAPP{alpha}695; open triangle, sAPP{alpha}770). C, fluorescence spectral center of mass in the presence of increasing concentrations of urea (filled circles, sAPP{alpha}695; filled triangles, sAPP{alpha}770). Complete unfolding was obtained by incubation in the presence of 7 M urea and 1 mM dithiothreitol at 100 °C for 30 min (cross-hair, sAPP{alpha}695; diamond, sAPP{alpha}770). Refolded samples were obtained by removal of urea using a desalting column (open circle, sAPP{alpha}695; open triangle, sAPP{alpha}770).

 

The equilibrium unfolding of sAPP{alpha}695 and sAPP{alpha}770 by urea is shown in Fig. 2C. Interestingly, for both proteins unfolding by urea appeared to follow a two-state transition (albeit a highly noncooperative one) between the native and unfolded states, without significantly populated partially unfolded intermediates. Complete unfolding of sAPP could not be achieved even at 7.6 M urea, and full reversibility of the transition was also observed upon removal of urea from previously unfolded samples (Fig. 2C).

Bis-ANS Binding Studies—The polarity-sensitive fluorescent probe, bis-ANS, was used in order to investigate the nature of the conformational intermediates stabilized in the presence of low concentrations of GdnHCl. Bis-ANS binds to exposed hydrophobic surfaces in partially folded intermediates with much higher affinity than to native or completely unfolded proteins (16–19), resulting in a marked increase in fluorescence emission compared with the emission of free bis-ANS in aqueous solution. Interestingly, we found that bis-ANS binds to native sAPP{alpha}695 (Fig. 3A) and sAPP{alpha}770 (data not shown). The addition of 0.5 M GdnHCl led to an increase in fluorescence emission compared with the native protein, indicating additional exposure of hydrophobic domains. As expected, the addition of 5 M GdnHCl (a concentration that causes extensive unfolding of sAPP; Fig. 2B) completely abolished the binding of bis-ANS to sAPP. Fig. 3B shows the fluorescence emission of bis-ANS in the presence of both sAPP{alpha} isoforms and denaturants (GdnHCl or urea), normalized by the fluorescence emission of bis-ANS in the presence of native sAPP{alpha}. For both isoforms, there was an initial sharp increase in bis-ANS fluorescence emission up to ~0.5 M GdnHCl. A gradual decrease in bis-ANS fluorescence emission was observed at higher GdnHCl concentrations. By contrast, a completely different pattern was observed when urea was used as a denaturant. In that case, no increase in bis-ANS binding was observed at low urea concentrations (Fig. 3, A and B), indicating that urea did not stabilize partially folded intermediates of sAPP{alpha} exhibiting exposed hydrophobic regions. Higher urea concentrations also led to a decrease in bis-ANS fluorescence emission (Fig. 3A).



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  		FIG. 3.
Unfolding of sAPP{alpha}695 and sAPP{alpha}770 monitored by bis-ANS fluorescence. sAPP{alpha} (0.25 µM) was diluted in 50 mM Tris-Cl (pH 7.4) in the presence of 10 µM bis-ANS. A, fluorescence emission spectra of bis-ANS in the presence of sAPP{alpha}695 under native conditions (———), in the presence of 0.5 M GdnHCl (–––), 0.5 M urea (····), 5 M GdnHCl (–·–·–), or 7 M urea (–··–··–). Very similar results were obtained with sAPP{alpha}770. B, sAPP{alpha}695 or sAPP{alpha}770 (closed and open symbols, respectively) was incubated overnight in the presence of bis-ANS and increasing concentrations of GdnHCl (circles) or urea (triangles). Data were normalized by the fluorescence emission obtained for bis-ANS in the presence of native sAPP{alpha}695 or sAPP{alpha}770.

 

Circular Dichroism Analysis—The secondary structures of sAPP{alpha}695 and sAPP{alpha}770 in the presence of denaturants were analyzed by circular dichroism. Fig. 4A shows CD spectra of native sAPP{alpha}695, in the presence of intermediate concentrations of denaturant (2.2 M GdnHCl or 2.9 M urea) or in the presence of 6 M GdnHCl or 7.6 M urea. At intermediate concentrations of denaturants, marked decreases in secondary structure of sAPP{alpha} were indicated by the decrease in ellipticity at both 208 and 222 nm, and the secondary structure content was almost completely lost in the presence of high concentrations of both denaturants. Circular dichroism spectra of sAPP{alpha}770 showed essentially the same behavior as observed for sAPP{alpha}695 (data not shown). Fig. 4B shows a plot of the molar residual ellipticity at 222 nm for both isoforms in the presence of increasing concentrations of GdnHCl. For both isoforms, the secondary structures remained essentially unaffected up to 1 M GdnHCl. Between 1 and 3 M GdnHCl, there was a sharp loss in secondary structure content, and a plateau indicative of some residual secondary structure content was achieved at higher concentrations of GdnHCl. Fig. 4C shows that the secondary structures of both sAPP isoforms were preserved up to ~1 M urea, with most changes in CD taking place between 2 and 4 M urea. CD data also indicated preservation of some residual secondary structure in both isoforms at high (7.6 M) urea concentrations.



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  		FIG. 4.
Unfolding of sAPP{alpha}695 or sAPP{alpha}770 monitored by circular dichroism. A, CD spectra of native sAPP{alpha}695 (———) or in the presence of 2.2 M GdnHCl (– – – –), 2.93 M urea (— —), 6.0 M GdnHCl (····), or 7.6 M urea (–·–·–). For the spectra up to 2.93 M denaturant, sAPP{alpha}695 (3.5 µM) was diluted in 50 mM Tris-Cl, pH 7.4, and incubated overnight at 8 °C with GdnHCl or urea, and spectra were measured in a 0.02-cm path length cell. Above 2.93 M denaturant, sAPP{alpha}695 (0.7 µM) was diluted in 50 mM Tris-Cl, pH 7.4, and incubated overnight with GdnHCl or urea, and spectra were measured in a 0.1-cm path length cell. B, molar residual ellipticity at 222 nm of sAPP{alpha}695 (circles) or sAPP{alpha}770 (triangles) in the presence of increasing concentrations of GdnHCl. sAPP{alpha}695 (0.70 µM) and sAPP{alpha}770 (1.26 µM) were diluted in 50 mM Tris-Cl, pH 7.4, and incubated overnight at 8 °C in the presence of different GdnHCl concentrations. C, molar residual ellipticity at 222 nm in the presence of urea (same symbols as in B). The concentrations of sAPP{alpha}695 and sAPP{alpha}770 were 1.38 and 1.26 µM, respectively, and samples were incubated overnight at 8 °C in the presence of increasing urea concentrations.

 

UV Absorption Spectroscopy—As an additional probe of the unfolding transitions of sAPP{alpha}, the near UV absorption spectra of sAPP{alpha}695 in the presence of different concentrations of denaturants were measured. In the presence of denaturants, the absorption of aromatic amino acid residues in proteins decreases, and characteristic difference peaks may be observed (13, 20, 21). The difference spectra of sAPP{alpha}695 obtained in the presence of GdnHCl and urea contained a principal peak at 286 nm and subsidiary peaks at 279 and 292–293 nm (Fig. 5, A and B). The main peak at 286 nm was used to monitor the unfolding of sAPP{alpha}695. The main transition occurs between 1 and 2 M GdnHCl or between 1.5 and 4.5 M urea, with a plateau reached at higher concentrations of denaturant (Fig. 5C). Similar results were obtained for the denaturation of sAPP{alpha}770 by GdnHCl (data not shown). Thus, the unfolding of sAPP{alpha} as monitored by near UV absorption spectroscopy roughly parallels the loss of secondary structure of the protein as revealed by CD (Fig. 4C).



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  		FIG. 5.
Near UV Absorption spectroscopy of sAPP{alpha}695. A, difference absorption spectra of sAPP{alpha}695 in the presence of 1.0 M (solid line), 1.5 M (long dashed line), 2.0 M (short dashed line), 4.0 M (dotted line), or 6.0 M GdnHCl (dotted and dashed line). sAPP{alpha}695 was diluted in 50 mM Tris-Cl, pH 7.4, with the appropriate concentrations of GdnHCl for a final protein concentration of 7.17 µM. B, difference absorption spectra of sAPP{alpha}695 in the presence of 1.5 M (solid line), 3.0 M (dashed line), 4.5 M (dotted line), or 8.0 M urea (dotted and dashed line). sAPP{alpha}695 was diluted in 50 mM Tris-Cl, pH 7.4, with the appropriate concentrations of urea for a final protein concentration of 7.17 µM. C, differential absorption (at 286 nm) of sAPP{alpha}695 in the presence of increasing concentrations of GdnHCl (circles) or urea (triangles).

 

SAXS Measurements and Analysis—SAXS is a sensitive technique for analyzing the global shape and dimensions of macromolecules in solution. We have used high resolution synchrotron radiation SAXS measurements to analyze the unfolding transitions of both sAPP isoforms. Fig. 6 (A and B) shows the raw intensity curves of sAPP{alpha}695 and sAPP{alpha}770 for the whole range of q measured after subtraction of the buffer signal. Scattering curves for both isoforms in the presence of 0.25 M GdnHCl, 2.0 M GdnHCl, and 6.0 M urea were also acquired and treated in the same way. In Fig. 6 (C and D), the curves corresponding to the native protein (black symbols) are shown together with the curves corresponding to the samples containing denaturants in the q region up to 0.3 Å–1. For the latter curves, data obtained at higher q values were not employed, because the statistical errors became much larger than for the pure protein. All curves were fitted and extrapolated to zero angle using the GNOM program (14) (continuous lines). From the zero angle scattering values, I(0), the molecular masses of sAPP{alpha}695 and sAPP{alpha}770 were calculated as 67 and 74 kDa, respectively, using albumin as a secondary standard. The ~7-kDa difference in molecular mass agrees well with the fact that sAPP{alpha}770 contains 75 additional amino acids as compared with sAPP{alpha}695 (5). For both isoforms, the scattering in 0.25 M GdnHCl (red symbols) is quite similar to that of the native protein. The slightly lower extrapolated I(0) (which is more noteworthy in the data for sAPP{alpha}695) may indicate a lower contrast between the protein and the solvent. In the presence of higher denaturant concentrations, the scattering curves of both sAPP{alpha} isoforms change considerably, indicating significant changes in particle shape and dimensions. For sAPP{alpha}695 in the presence of 2 M GdnHCl (green symbols), the scattering intensity begins at a lower I(0) value and crosses the native curve at ~0.12 Å–1 (Fig. 6C). In the presence of 6 M urea (blue symbols), the scattering function is similar to that observed in the presence of 2 M GdnHCl, indicating similarities in the denaturation process. Scattering data of sAPP{alpha}695 in the presence of 6 M GdnHCl could not be interpreted because of the very high x-ray absorption by the buffer (data not shown). For the sAPP{alpha}770 isoform, the behavior of the intensity curves as a function of added denaturant is somewhat different (Fig. 6D). These curves also show different values of I(0) but do not exhibit marked differences in the intensity at higher q values.



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  		FIG. 6.
Upper row, SAXS intensity data for pure sAPP{alpha}695 (A) and sAPP{alpha}770 (B) in the whole q range measured. Data measured at concentrations up to 12.5 mg/ml (sAPP{alpha}695) or 9.6 mg/ml (sAPP{alpha}770) were found to agree perfectly and used to construct the curve over the complete q range. The experimental intensities and statistical errors are plotted against q (equal to 4{pi}*sin({theta})/{lambda}). Lower row, sAPP{alpha}695 (C) and sAPP{alpha}770 (D) scattering curves in the presence of denaturants. Concentrations of sAPP{alpha}695 and sAPP{alpha}770 were 3.6 and 3.2 mg/ml, respectively. Black symbols, native sAPP{alpha}. Red symbols, sAPP{alpha} in the presence of 0.25 M GdnHCl. Green symbols, sAPP{alpha} in the presence of 2 M GdnHCl. Blue symbols, sAPP{alpha} in the presence of 6.0 M urea. Solid lines, data fitted and extrapolated to zero angle using the GNOM program (15).

 

The radii of gyration (Rg) for both sAPP{alpha} isoforms in their native states and in the presence of denaturants were calculated with the GNOM program (Table I). The Rg of native sAPP{alpha}695 is slightly higher than the previously reported value (12). We consider this a more reliable value, due to the much higher resolution of the present data. In accordance with the expectations, the Rg of native sAPP{alpha}770 is 4 Å larger than that of native sAPP{alpha}695, indicating a somewhat larger molecular dimension. The addition of up to 2 M GdnHCl caused hardly any change in Rg for sAPP{alpha}695 and an increase of <2 Å in Rg for sAPP{alpha}770. On the other hand, the addition of 6 M urea caused an increase in Rg of 9.5 Å for sAPP{alpha}695 and 11.3 Å for sAPP{alpha}770.


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  		TABLE I
Radii of gyration of sAPP{alpha}695 and sAPP{alpha}770

Values were calculated from SAXS data using the GNOM program (14). Measures at 1.6 mg/ml (for pure sAPP{alpha}) and 3.2 mg/ml did not give different Rg values.

 

The radius of gyration reflects the overall size of the protein particle, and, thus, many changes in the structure of a protein may not affect its radius of gyration (22). For a more sensitive analysis of the effects of chemical denaturants on the global compactness of sAPP{alpha} isoforms, we have analyzed the SAXS data in terms of the Kratky plot (23). For a system with compact shape and sharp phase boundaries, this plot shows a well defined curve with an initial upward portion followed by a descending curve (24). On the other hand, the curve for a polymer in an extended or random coil conformation shows a characteristic plateau and rises at higher angles (25, 26). Therefore, this plot has been shown to be quite useful to monitor the unfolding of proteins (e.g. see Refs. 27 and 28). It has also been shown that the form of the Kratky plot depends more on the preservation of a compact core than on differences in secondary structure (29).

Both native sAPP{alpha} isoforms showed well defined maxima at low q in the corresponding Kratky plots (Fig. 7, black symbols and lines), indicating that they are relatively compact particles. However, it is noteworthy that the scattering intensities do not fall as steeply at higher q as would be expected for a sharp phase boundary (i.e. a particle without loose parts) (see Refs. 27 and 29), suggesting a certain extent of structural looseness of sAPP{alpha} even in the native state. This is in line with our recent report of the possible presence of >30% nonstandard secondary structure in native sAPP{alpha}695 (12).



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  		FIG. 7.
Kratky plots for sAPP{alpha}695 (A) and sAPP{alpha}770 (B). Black symbols, native sAPP{alpha}. Red symbols, sAPP{alpha} in the presence of 0.25 M GdnHCl. Green symbols, sAPP{alpha} in the presence of 2 M GdnHCl. Blue symbols, sAPP{alpha} in the presence of 6.0 M urea. Solid lines, Kratky plots (27) of data fit obtained using the GNOM program (15).

 

In the presence of 0.25 M GdnHCl (red symbols), the maxima of the curves for both isoforms are shifted to higher q, but the curves still exhibit a clear descent at higher q values. By contrast, in the presence of 2 M GdnHCl (green symbols) or 6 M urea (blue symbols), the curves do not show any maxima but instead an initial plateau up to 0.09 Å–1. This type of plateau is typical of the intermediate q region for Gaussian coils (26). Furthermore, at higher q the curves do not descend but rather rise, as typically exhibited by elongated chains (26). Thus, the Kratky plots of the scattering data of sAPP{alpha} in the presence of 2 M GdnHCl or 6 M urea suggest that both isoforms are largely unfolded. The upward curvature in the Kratky plots in the high q range is more pronounced in the case of sAPP{alpha}695, indicating that it may adopt a random coil conformation approaching a state of complete denaturation. On the other hand, the smoother increase in the Kratky curves of sAPP{alpha}770 indicates that this isoform does not completely denature in the presence of the same concentrations of denaturants. Since the main differences in the scattering curves occur for q > 0.1 Å–1, which corresponds to molecular details on the order of ~30 Å, our results indicate that for the sAPP770 isoform, structural features of that size are not completely denatured even with the loss of the general protein conformation. On the other hand, in the presence of 6 M urea, sAPP{alpha}770 exhibits a loss of structure similar to that of the other isoform, assuming a random coil conformation, indicated by the rising intensity in the Kratky plot.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
The amyloid precursor protein has been known for about 15 years, and its relevance to the physiopathology of Alzheimer's disease is well established. However, relatively little is still known about the normal physiological roles and binding partners of APP, and even less is known about its structure. In order to provide insight into the roles and functional mechanisms of APP, a more detailed structural characterization of this protein is clearly needed. In this work, the folding and stability of sAPP{alpha} were investigated using different approaches and are related to recently elucidated structural features of the protein.

The intrinsic fluorescence data (Fig. 2A) show that native sAPP{alpha} has an emission peak at relatively high wavelengths (~337 nm), suggesting that, on average, its eight tryptophan residues are partly exposed to the aqueous medium. Its far-UV CD spectrum indicates a substantial amount of helical structure (~35%) but also a significant amount (>30%) of nonstandard secondary structure elements (Fig. 4A) (see also Ref. 12). APP can be completely and reversibly unfolded by treatment with 6 M GdnHCl and 1 mM dithiothreitol at 100 °C or by incubation in the presence of 6.5 M GdnHCl at room temperature (Fig. 2B). In the unfolded state, sAPP{alpha} has a maximum fluorescence emission at ~350 nm (Fig. 2A), typical of tryptophan residues that are fully exposed to the polar solvent. In agreement with the position of the fluorescence peak, the extinction coefficient of unfolded sAPP{alpha}695 (58,470 M–1 cm–1, data not shown) is close to the value estimated for its 14 tyrosine and 7 tryptophan residues in a polar medium (57,960 M–1 cm–1) (see Ref. 20). Furthermore, in the unfolded state, APP does not bind bis-ANS (Fig. 3A) and loses >85% of its secondary structure, as monitored by CD (Fig. 4, A and B). The equilibrium unfolding of sAPP{alpha} was followed by measuring the red shift in the intrinsic fluorescence center of mass (Fig. 2B). Interestingly, in the presence of GdnHCl, the equilibrium unfolding does not follow a simple two-state (native {leftrightarrow} unfolded) transition. Instead, the data indicate the presence of intermediate partially folded states at ~1 and ~3 M GdnHCl (Fig. 2B).

Analysis of the bis-ANS fluorescence data shows a rise in bis-ANS binding to sAPP{alpha} in the presence of 0.125–0.5 M GdnHCl (Fig. 3B), indicating increased exposure of organized hydrophobic patches in the protein (16–19). We therefore conclude that in the presence of low concentrations of GdnHCl (<0.5 M) sAPP{alpha} undergoes conformational changes that lead to higher bis-ANS binding, whereas the average environment of its tryptophan residues changes only slightly. Furthermore, at these low concentrations of GdnHCl, the secondary structure of sAPP{alpha} is largely unaffected (Fig. 4B), whereas the Kratky plot of SAXS data indicates an incipient loosening of the compact core of the protein, as indicated by a slight shift of the peak to higher q values (Fig. 7).

The partially folded intermediate state populated in the presence of 3 M GdnHCl no longer exhibits exposed organized hydrophobic domains, as judged by the loss of bis-ANS binding (Fig. 3B). On the other hand, most of the loss of secondary structure of APP takes place between 1 and 2 M GdnHCl (Fig. 4B) as well as most of the changes in near UV absorption (Fig. 5, A and C). The intermediate state observed at 3 M GdnHCl exhibits <30% of the native secondary structure of sAPP{alpha} (Fig. 4B). In agreement with the CD data, the Kratky plot shows that sAPP{alpha} at 2 M GdnHCl has largely lost its compact core, as indicated by the absence of a peak at low q values (Fig. 7) (27, 29). Interestingly, the radius of gyration of sAPP{alpha} in the presence of 2 M GdnHCl is similar to that found in the absence of denaturant (Table I). This, however, should not be considered too surprising, since it has been pointed out that distinct conformations of a given molecule with very different compactness and, accordingly, very different Kratky plots can have the same average radii of gyration (22). In conclusion, in the presence of 3 M GdnHCl sAPP{alpha} has a low secondary structure content and has largely lost its compact core, but part of its tryptophan residues encounter a different excited state environment from that of the aqueous medium.Go



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In contrast with the GdnHCl data, very different equilibrium unfolding curves were obtained for sAPP{alpha} in the presence of urea (Fig. 2C). Urea did not stabilize any partially folded state of APP, and broad, smooth transitions from the native to the unfolded states were observed for both sAPP isoforms. Furthermore, no increase in bis-ANS binding to sAPP{alpha} was observed in the presence of urea (Fig. 3B). It is interesting to note, however, that the c1/2 values obtained from intrinsic fluorescence, on the one hand, and UV absorption and CD changes, on the other hand, were ~5.5 and ~3 M urea, respectively. This indicates that the changes in secondary structure, excited state environment of tryptophan residues and overall conformation of sAPP{alpha} did not occur in parallel as a function of increasing concentrations of urea. Furthermore, it should be noted that the conformational changes induced by urea took place over a very broad concentration range, with a highly noncooperative transition between 1 and 8 M urea (Fig. 2C). Taken together, these observations indicate that the unfolding of sAPP{alpha} by urea is not a true two-state transition but rather involves partially folded intermediates, which, however, are not sufficiently populated to be detected by spectroscopic techniques.

It is well known that the concentration of GdnHCl necessary to unfold a protein is generally different from the concentration of urea necessary to achieve the same degree of unfolding. Usually, the effects of a given concentration of GdnHCl are stronger than those of the same concentration of urea. Since GdnHCl dissociates into the chaotropic guanidinium cation and the chloride anion, whereas urea is a neutral molecule, an ionic force effect might be held responsible for the greater unfolding strength of GdnHCl (30). To test the hypothesis that the different efficacies of GdnHCl and urea on the unfolding of sAPP{alpha} were due to an ionic strength effect, we carried out urea unfolding experiments in the presence of 0.5 M NaCl. This concentration of NaCl was chosen to mimic the ionic strength of a 0.5 M GdnHCl solution, at which the first partially folded intermediate state of sAPP{alpha} was observed (Figs. 2B and 3B). The unfolding curve in the presence of urea was unaltered by the addition of 0.5 M NaCl (data not shown), excluding the possibility that the stabilization of partially folded states of sAPP{alpha} by GdnHCl could be due to a simple ionic strength effect. This result also shows that the stabilization of the intermediate states is not due to an effect of the chloride anion. It has been pointed out that the different effects of GdnHCl and urea on protein stability cannot be entirely explained by ionic strength effects (31, 32). Thus, the effect of GdnHCl on the stability of sAPP{alpha} intermediates seems to be due to more specific interactions of the guanidinium cation with the protein.

SAXS measurements in the presence of high concentrations of GdnHCl (>2 M) were not possible due to the high x-ray absorption by the chloride anions in the solvent. However, data measured in the presence of 6 M urea showed that the radius of gyration of sAPP{alpha} increased by 22% (sAPP{alpha}695) or 24% (sAPP{alpha}770). According to the SAXS results, there are some differences between the denaturation processes of the two isoforms. Still, in both cases, the presence of a low concentration of GdnHCl induces a subtle conformational change without a rise in the Kratky plot at higher angles (Fig. 7), indicating a fair preservation of the globular nature of the protein and some loss of compactness.

In conclusion, we have shown the existence of at least two partially folded intermediates in the equilibrium unfolding of sAPP{alpha} by GdnHCl. These intermediates may represent the folding pathway of APP, with successive intermediates each having more native-like structure than their predecessors. Thus, the folding pathway of sAPP may be represented by the following minimal scheme.

Whereas I2 (observed at ~3 M GdnHCl) has nonnative secondary and tertiary structures, the I1 intermediate state (observed at <1 M GdnHCl) has native-like secondary structure, and its tryptophan residues experience an average environment similar to that of the tryptophan residues in the native state N. However, I1 exposes much more hydrophobic surface to the solvent than N. This may represent a rearrangement of two or more APP domains in the presence of low GdnHCl concentrations, which is confirmed by the SAXS results. It was suggested upon the discovery of the APP gene that the extracellular portion of the protein may consist of several distinct domains (4) (see also Fig. 1). Specifically, the KPI domain present in the longer isoforms of sAPP{alpha} appears to behave as an autonomous folding unit, as indicated by the fact that it forms crystals suitable for x-ray diffraction (33). The same considerations apply to the N-terminal domain (34) and the copper-binding domain (35). Other suggested domains of sAPP{alpha} include the zinc-binding domain and the heparin-binding domain (1). Of special interest are the highly acidic region located N-terminally to the KPI domain and the region of very low homology located N-terminally to the A{beta} sequence. Both of these domains may not contain standard secondary structure elements (12, 36). Therefore, the stepwise unfolding of the two sAPP{alpha} isoforms via partially folded intermediate states described here may correspond to the rearrangement and unfolding of different structural domains (i.e. relatively independent folding units) present in the extracellular portion of APP. For example, the presence of the KPI domain in sAPP{alpha}770, but not in sAPP{alpha}695, may contribute to the different patterns of unfolding of the two isoforms revealed in the Kratky plots (Fig. 7).

The fact that the first partially folded intermediate state of sAPP{alpha} is stabilized at quite low concentrations of GdnHCl (0.125–0.5 M) suggests that the native state of this protein is relatively unstable. Electrostatic or other types of interactions between the guanidinium cation and protein groups stabilize the intermediate state relative to the native state of sAPP{alpha}. It is tempting to speculate that interactions of the extracellular domain of APP with various types of ligands or exposure to different local environments in the extracellular medium may induce conformational changes to states similar to the I1 state stabilized at low concentrations of GdnHCl. Among the different physical microenvironments encountered by APP during its cellular lifetime are 1) the endoplasmic reticulum/Golgi apparatus, where immature transmembrane APP may already be cleaved by {beta}- and {gamma}-secretase (37); 2) cholesterol- and glycolipid-rich domains in the trans-Golgi network, secretory vesicles, and plasma membrane, where APP co-localizes with {alpha}- and/or {beta}-secretase and undergoes cleavage (38–40); and 3) the acidic interior of endosomes following internalization (41). Furthermore, after cleavage by {alpha}- or {beta}-secretase, the secreted extracellular domain leaves the chemical and physical neighborhood of the membrane to interact with multiple binding partners in the extracellular matrix (42). Finally, the secreted extracellular domain may also bind to distinct patches on the plasma membrane of other cells (43). One or more of these different environments may induce a domain rearrangement in the extracellular domain of APP similar to that induced by low concentrations of GdnHCl (Fig. 3). In this regard, it is interesting to note that sAPP{alpha} has been shown to be monomeric over a wide concentration range (see Table I and Ref. 12), whereas a tendency to dimerize has been proposed for both the isolated N-terminal half of sAPP{alpha} and for transmembrane APP (44). A region of nonstandard secondary structure unique to APP, as compared with its homologues, has been proposed to lie next to the membrane (12, 36). The structural rearrangement of sAPP{alpha} may involve a bending motion and/or oligomerization in response to a physiological signal, as recently reported for the activation of integrins (45).

Alternatively, or in conjunction with the influence of the physicochemical microenvironment, specific binding partners may induce structural changes in APP. Indeed, a number of different extracellular molecules have been proposed to bind APP (1). Thus, the inherent relatively low stability of APP may endow this protein with sufficient conformational plasticity to allow it to adapt to different binding partners, explaining in part the many different physiological functions proposed for APP.

Although APP is the best studied member of the APP superfamily and of special relevance because of its involvement in Alzheimer's disease, the member of the family most essential for normal development may be the APP-like protein 2 (APLP2). Despite conflicting data on the viability of homozygous APLP2 knockout mice, there is agreement on the fact that APLP2 knockouts have severer consequences than APP knockouts (46, 47). Functions proposed for APLP2 include specific DNA binding and chromosome segregation in the metaphase nucleus (47); binding and regulation of a major histocompatibility complex type I allele in the endoplasmic reticulum, in the Golgi apparatus, and on the cell surface (48); and epithelial wound healing (49). In view of the high homology between APP and APLP2 (12), the considerations on the flexibility and plasticity of the native state of APP raised above may also be relevant to account for the ability of APLP2 to fulfill such diverse physiological roles.


   FOOTNOTES
 
* This work was supported by grants from the John Simon Guggenheim Memorial Foundation, Howard Hughes Medical Institute, Conselho Nacional de Desenvolvimento Científico e Tecnológico, Fundação de Amparo à Pesquisa do Estado do Rio de Janeiro, and Financiadora de Estudos e Projetos (to S. T. F.) and by Fundação de Amparo à Pesquisa do Estado de São Paulo and Laboratório Nacional de Luz Síncrotron. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

|| A Howard Hughes Medical Institute International Scholar. To whom correspondence should be addressed. Tel./Fax: 5521-2562-6789; E-mail: ferreira{at}bioqmed.ufrj.br.

1 The abbreviations used are: A{beta}, amyloid {beta}; APP, amyloid precursor protein; bis-ANS, 4–4'-dianilino-1,1'binaphthyl-5,5'-disulfonic acid; GdnHCl, guanidine hydrochloride; KPI, Kunitz-type inhibitory; sAPP{alpha}, soluble fragment of APP released by {alpha}-secretase; SAXS, small-angle X-ray scattering; APLP2, APP-like protein 2. Back

2 C. L. P. Oliveira, unpublished observations. Back



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TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
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