Ionic self-complementarity induces amyloid-like fibril formation in an isolated domain of a plant copper metallochaperone protein
- Helena Mira†1,
- Marçal Vilar†1,
- Vicent Esteve1,
- Marc Martinell2,
- Marcelo J Kogan2,
- Ernest Giralt2, 3,
- David Salom4, 6,
- Ismael Mingarro1,
- Lola Peñarrubia1 and
- Enrique Pérez-Payá1, 5Email author
© Mira et al; licensee BioMed Central Ltd. 2004
Received: 18 February 2004
Accepted: 04 June 2004
Published: 04 June 2004
Arabidopsis thaliana copper metallochaperone CCH is a functional homologue of yeast antioxidant ATX1, involved in cytosolic copper transport. In higher plants, CCH has to be transported to specialised cells through plasmodesmata, being the only metallochaperone reported to date that leaves the cell where it is synthesised. CCH has two different domains, the N-terminal domain conserved among other copper-metallochaperones and a C-terminal domain absent in all the identified non-plant metallochaperones. The aim of the present study was the biochemical and biophysical characterisation of the C-terminal domain of the copper metallochaperone CCH.
The conformational behaviour of the isolated C-domain in solution is complex and implies the adoption of mixed conformations in different environments. The ionic self-complementary peptide KTEAETKTEAKVDAKADVE, derived from the C-domain of CCH, adopts and extended conformation in solution with a high content in β-sheet structure that induces a pH-dependent fibril formation. Freeze drying electron microscopy studies revealed the existence of well ordered amyloid-like fibrils in preparations from both the C-domain and its derivative peptide.
A number of proteins related with copper homeostasis have a high tendency to form fibrils. The determinants for fibril formation, as well as the possible physiological role are not fully understood. Here we show that the plant exclusive C-domain of the copper metallochaperone CCH has conformational plasticity and forms fibrils at defined experimental conditions. The putative influence of these properties with plant copper delivery will be addressed in the future.
Apparently natural β-sheet proteins seem to avoid aggregation by disfavouring solvent exposed sequences that could drive intermolecular aggregation . In fact, most of the natural proteins forming fibrillar aggregates are related to a range of human metabolic disorders . Among the aggregation processes, the amyloidosis is a group of protein misfolding disorders characterized by the formation of fibrils . Although most of these processes have been described in humans, other organisms also present examples of proteins forming amyloid fibrils, such as monellin from higher plants [3, 4]. However, the physiological relevance of the aggregation processes remains obscure. A number of these amyloid forming proteins are involved in copper homeostasis, and oxidative damage induced by copper may play a role in the pathogenesis of neurodegenerative conditions. β-amyloid (Aβ), implicated in the pathogenesis of Alzheimer's disease, forms an oligomeric complex that binds Cu2+ with very high affinity forming an allosterically cooperative Cu2+-coordination site that resembles superoxide dismutase 1 (SOD) . Aggregation of SOD has also been related to familial amyotrophic lateral sclerosis (ALS)  and copper-bound prion protein (PrP) [7, 8] shows superoxide dismutase activity .
Selection of a 19-mer peptide model derived from the C-domain of CCH
Since the C-domain showed a complex conformational behaviour in solution, we looked for a simplified peptide model that could facilitate the study of the apparent secondary structure plasticity of the CCH C-domain. We have focused our attention into the sequence KTEAETKTEAKVDAKADVE (amino acids 90 to 108 – Fig. 1A). Then, peptide-1 is an N-t acetylated, C-t amidated synthetic peptide with such amino acid sequence where a tyrosine residue was placed at the N-terminal end to facilitate quantification by UV spectroscopy (Fig. 1B). Peptide-1 contains a segment of alternating hydrophobic and hydrophilic residues and a striking overall charge distribution. These two properties have been early described as determinants for secondary structure plasticity [15, 16, 22]. In fact, this sequence was predicted to fold as an α-helix by secondary structure prediction methods using the PHD software (EMBL, Heidelberg, Germany) applied to both, to the amino acid sequence of the whole CCH and to that of the C-domain. The putative α-helix adopted by peptide-1 would show charge segregation due to the relative orientation of positively and negatively charged residues (Fig. 1C). However, when the amino acid sequence of peptide-1 is analyzed for secondary structure prediction using the program AGADIR  the predicted helical content is extremely low. Furthermore, such an amino acid sequence could be arranged into an amphipathic β-sheet conformation (Fig. 1D) that would expose a hydrophobic face and a highly charged hydrophilic face with three pairs Glu-Lys and one pair Asp-Lys. Then, the peptide sequence could be intra- or inter-molecularly stabilized by means of ionic self-complementarity that would facilitate the formation of a macromolecular β-pleated-sheet complex.
Conformational analysis of peptide-1
Amino acid sequences of synthetic peptide-1 and analogues
Altered electrophoretic mobilitya
The conformational behaviour of peptide-1 in solution at different pHs was analyzed by CD and NMR spectroscopies. At neutral pH and 10°C (Fig. 4B), the CD spectrum of peptide-1 is similar to that obtained for the whole C-domain and it is characterized by a strong negative ellipticity band with a minimum at 198 nm characteristic of an extended β-sheet secondary structure. As the temperature increased (from 10–70°C – insert in Fig. 4B), the spectra showed less intense bands suggesting a decrease in secondary structure. At basic pH values, the negative band at 198 nm of the far-UV CD spectrum obtained at neutral pH was slightly increased (Fig. 4B). However, at acidic pH values, the CD analysis suggested a conformational transition towards the stabilization of a β-sheet secondary structure that produces a canonical β-sheet CD spectrum with a minimum located at 215 nm (Fig. 4B). 1H NMR monodimensional spectra of a 50 μM solution of peptide-1 (i.e. the same concentration used in CD) were acquired in water at different pH values and an increase of dispersion of the signals in the aliphatic region was observed when the pH was changed from 2.7 to 7 (Fig. 4C). No major differences were observed in other regions of the spectra. In order to proceed to chemical shift assignments the NMR behaviour of peptide-1 at higher concentrations was explored. At 3 mM in water with pH adjusted to 2.5 with HCl peptide-1 was soluble and the complete assignment of the amide resonances was achieved by analysis of COSY and TOCSY spectra. When the pH of the 3 mM solution of peptide-1 was raised to 4.5 a reversible transition to gel state was visible on the NMR glass tube, whereas at pH 7 the peptide aggregation was irreversible. At 1 mM pH 5.7 the complete assignment of the amide resonances was also achieved. Secondary α H chemical shift data suggest the putative presence of transient secondary structure elements although the spectra are strongly influenced by peptide conformations without significant persistent secondary structure (data not shown). These results do not correlate, apparently, with those obtained when the peptide-1 was analysed by CD spectroscopy as a function of pH where different types of characteristic β-sheet CD spectra were obtained (Fig. 4B). However, as previously described by Gross et al.  in their elegant study on the aggregation of peptides derived from the bacterial cold shock protein CspB, it must be taken into account that in conventional solution NMR we detect only the low molecular weight species, whereas in CD we can also detect high molecular weight soluble aggregates with a β-sheet structure.
Amyloid-like fibril formation of peptide-1
The NMR and CD data suggest that peptide-1 can exist in solution as a complex mixture of conformations that could extend from low molecular weight and unstructured species to large oligomers with a predominant secondary structure in β-sheet. This prompted us to expand our studies by using tools able to diagnose fibril formation.
In previous work, we have shown that the Arabidopsis thaliana copper metallochaperone CCH, the functional homologue of yeast Atx1, contains an extra C-terminal domain with unusual structural properties. This domain has a folding pathway independent from the N-terminal domain and it confers to the whole CCH protein an altered electrophoretic mobility in SDS-PAGE . In the present study, we have further characterized the CCH C-domain and delimited a peptide that accounts for most of its structural properties such as its high tendency to form oligomers. We also show that such peptide oligomerizes to form amyloid-like fibrils and that β-strand structure in solution and self-complementary attractive electrostatic interactions could be a requirement for fibrillogenesis.
The recombinant C-domain adopts an extended (beta) structure in solution at acidic, neutral and basic pH that induces the formation of fibril structures as determined by TEM. Furthermore, as demonstrated by altered electrophoretic mobility  and analytical ultracentrifugation (Fig. 3) the C-domain shows tendency to participate in SDS-induced oligomerization events.
The Arabidopsis genome possesses an ATX1 homologue in addition to the plant exclusive CCH protein. It is reasonable to assume that the ATX1 homologue would perform the function of copper delivery to the secretory pathway transporter described in yeast . Since CCH has an exclusive C-domain, absent in the non-plant homologues and it probably reaches its location at the phloem sieve elements through plasmodesmata (the plant intercellular symplastic connections), we have postulated that the C-domain could accomplish this exclusive plant function . Extra C-domains have been also found in other plant cuprochaperones (Mira et al., unpublished results). In order to transport copper through plasmodesmata, higher plant copper metallochaperones could have been evolved the addition of new protein domains that could facilitate the cell-to-cell export mechanism. Although the mechanistic aspects of the process are not yet solved and no consensus sequences are present in the proteins demonstrated to cross plasmodesmata, several characteristics of the CCH C-domain points to a role in that process. The C-domain and peptide-1, have conformational flexibility and can be induced to populate different conformations in different membrane-mimetic media like SDS and TFE (Fig. 5) and at different pH values (Figs. 4B and 5B). Such a conformational flexibility could be relevant to allow a protein transport through plasmodesmata which contain membranous extension of the endoplasmic reticulum . The pH change at the phloem  could also serve to modulate the conformational steps required in the transport process. Moreover, it has been postulated that microtubules could also play a role in targeting proteins to plasmodesmata  and recently, the microtubules have been related to fibrillation processes. In fact, the formation of the neurodegenerative deposits by the fibrillar β-amyloid peptide is affected by the presence of stable microtubules  and α-synuclein, subjected to fibrillar aggregation, exploits its interaction with microtubules to address to a specific cellular location . Recently ATX1 mammalian homologues have been shown to play a role not only in copper delivery but in intracellular trafficking of the corresponding copper ATPases . These results uncover a complex function of cuprochaperones in membrane location of their targets. We cannot rule out the possibility of CCH playing a specific role in intracellular membrane trafficking where its C-terminal domain may have a role. In this sense, preliminary results using the two-hybrid technique indicate that the C-terminal domain of CCH modulates the interaction with the corresponding plant copper-transporting ATPases (Sancenón, V. and Peñarrubia, L., unpublished results).
A number of proteins related with copper homeostasis have a high tendency to form fibrils. The determinants for fibril formation, as well as the possible physiological role are not fully understood. It has been postulated that a problem associated to the interaction between copper binding proteins and biological membranes is the dangerous generation of H2O2 through Cu2+ reduction, which make cells more responsive to oxidative stress . Atx-like metallochaperones have a copper-binding motif and antioxidant properties [47, 48] related to the CCH N-domain , while the domain implicated in the fibrillation process is the C-domain. The putative reciprocal influence between these properties should be addressed in the whole protein.
Expression and purification of the C-domain of CCH
The recombinant C-domain of CCH was expressed and purified from Escherichia coli DH5α cells as previously reported .
Secondary structure predictions were determined using the PHD software (EMBL, Heidelberg, Germany) and the program AGADIR .
Peptides were synthesized by solid-phase multiple peptide synthesis  using Fmoc chemistry  and purified by reversed-phase high performance liquid chromatography (RP-HPLC) using a C18 column. Analytical RP-HPLC and laser desorption time-of-flight mass spectrometry were used to determined the purity and identity of the peptides.
Circular dichroism spectroscopy
All measurements were carried out on a Jasco J-810 CD spectropolarimeter, in conjunction with a Neslab RTE 110 waterbath and temperature controller. CD spectra were the average of a series of ten scans made at 0.2 nm intervals. CD spectra of the same buffer (or in the presence of trifluorethanol (TFE) or sodium dodecyl sulfate (SDS) as described in the Figure Legends) without peptide were used as baseline in all the experiments. For peptide-1 and analogues the concentration was determined by UV spectrophotometry using ε276,Tyr = 1450 M-1cm-1. The concentration of the C-domain was obtained by quantitative amino acid analysis.
Sedimentation equilibrium analysis of the CCH C-domain was performed using a Beckman XL-I analytical ultracentrifuge. Three solutions of the C-domain at concentrations of 0.9, 0.45 and 0.23 mg/mL were prepared in 50 mM Tris-HCl pH 8.0, containing 0.2 M NaCl and centrifugated at 50000 rpm. An additional solution of the C-domain at 0.5 mg/mL was also prepared in the presence of 1 mM SDS, and centrifuged at 45000 rpm, at 25°C. The equilibrium was determined when successive radial absorbance scans at the same speed were indistinguishable. Partial specific volumes and molecular weights were estimated for the C-domain using the software Sedinterp . The amino acid partial specific volumes were updated using the values reported by Kharakoz . For the C-domain the calculated values were 0.7404 mL/mg and 4936.6 Da. Solution density was estimated to be 1.00781 g/mL also using "Sedinterp". Data obtained by UV absorbance were analyzed by non-linear least squares curve fitting of radial concentration profiles using the Marquardt-Levenberg algorithm implemented in Igor Pro (Wavemetrics, Oswego, OR) with a user-defined function, as previously described . Sedimentation equilibrium analysis of peptide-1 was performed at the Analytical Ultracentrifugacion Facility, Centro de Investigaciones Biológicas, CSIC, Madrid.
NMR spectra were recorded using a Bruker AMX-500 (500 MHz 1H) NMR spectrometer. 1H NMR spectra were acquired using H20 10% D2O as solvent at 298 K. The pH of the solutions was adjusted with solutions of 10 mM HCl or 10 mM NaOH.
Congo red and thioflavine T binding analysis
For the Congo red (Merck) binding assay of fibril formation, absorption spectra of 10 μM solution of the dye at different pH values before and after addition of the peptide-1 solution (final concentration 50 μM) were recorded in a Cintra 10 e spectrometer (GBC Sci. Equip., Australia). For the thioflavine T (Aldrich) binding assay, fluorescence emission spectra of 10 μM solution (in Tris-HCl 25 mM pH 7.2 buffer) of the dye were measured before and after addition of the peptide-1 solution (final concentration 200 μM) on a Perkin-Elmer (Beaconsfield, UK.) LS-5B spectrofluorimeter at excitation wavelength of 440 nm. The bandwidths of excitation and emission were 5 nm.
Freeze fixation, freeze drying electron microscopy
Drops of recombinant C-domain or peptide-1 solutions (see text) were deposited over uncoated coverslips. Coverslips were cryofixed by projection against a copper block cooled by liquid nitrogen (-196°C) using a cryoblock (Reicher-Jung, Leica). The frozen samples were stored at -196°C in liquid nitrogen until subsequent use. Samples were freeze-dried at -90°C and coated with platinum and carbon using a freeze-etching unit (model BAF-060, BAL-TEC, Liechtenstein). A rotatory shadowing of the exposed surface was made by evaporating 1 nm platinum-carbon at an angle of six degrees above the horizontal, followed by 10 nm of carbon evaporated at an angle of ninety degrees. The replica was separated from the coverslip by immersion in concentrated hydrofluoric acid, washed twice in distilled water and digested with 5 % sodium hydrochloride for 5 – 10 minutes. Finally, the replicas were washed several times in distilled water, broken into small pieces and collected on Formvar-coated copper grids for electron microscopy. All electron micrographs were obtained using an electron microscope Hitachi 800 MT operating at 75 KV. Up to ten different samples were prepared by this procedure and the results were reproducible among all the samples assayed.
The molecular dynamics simulation was carried out at 300 K during 1000 ps at pH 7 and ε = 4r using CVFF (implemented in DISCOVER_3) as force field. The torsion angles of one unit of peptide-1 backbone were restrained to values between -180° and -60° for the φ angle and between 180° and 80° for the φ angle (typical β-sheet range in a Ramachandran plot). Two peptide-1 molecules in an antiparallel arrangement were selected to run the molecular dynamics simulation. This arrangement of the peptide molecules allows the putative formation of a maximised number of both salt bridges and hydrogen bonds.
This work was supported by grants BIO4-CT97-2086 (EU Biotechnology), SAF01-2811, BIO2002-1125 and BIO2002-2301 (MCyT and FEDER), and Generalitat de Catalunya (Grups Consolidats de Recerca and Centre de Referència en Biotecnologia). We are grateful to Dr. William DeGrado (Univ. Pennsylvania) for the use of the Beckman XL-I analytical ultracentrifuge. We thank Alicia García and Ana Giménez for excellent technical work.
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