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Ionic self-complementarity induces amyloid-like fibril formation in an isolated domain of a plant copper metallochaperone protein.

Mira H, Vilar M, Esteve V, Martinell M, Kogan MJ, Giralt E, Salom D, Mingarro I, Peñarrubia L, Pérez-Payá E - BMC Struct. Biol. (2004)

Bottom Line: 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.

View Article: PubMed Central - HTML - PubMed

Affiliation: Departament de Bioquímica i Biologia Molecular, Universitat de València, E-46100 Burjassot, València, Spain. helena.mira@uv.es

ABSTRACT

Background: 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.

Results: 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 beta-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.

Conclusion: 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.

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Hypothetical model for peptide-1 fibril formation based on a molecular dynamics simulation. (A) Initial structure with two units of peptide-1 arranged in a β-antiparallel disposition. An intermolecular distance of 4 Å within the complementary charged amino acid residues (between the charged atoms) was used. (B). Snapshot representative of the final part of the simulation where the energy of the system was stabilized. (Blue: hydrophobic residues, green: acidic residues; magenta: basic residues).
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Figure 8: Hypothetical model for peptide-1 fibril formation based on a molecular dynamics simulation. (A) Initial structure with two units of peptide-1 arranged in a β-antiparallel disposition. An intermolecular distance of 4 Å within the complementary charged amino acid residues (between the charged atoms) was used. (B). Snapshot representative of the final part of the simulation where the energy of the system was stabilized. (Blue: hydrophobic residues, green: acidic residues; magenta: basic residues).

Mentions: A synthetic peptide (peptide-1 – Fig. 1) derived from the C-domain was selected in order to facilitate the conformational study. Peptide-1 has most of the general sequence properties of the C-domain. It is characterised by a pattern of alternating hydrophobic and hydrophilic residues and by a striking overall charge distribution containing a 30% and 20% of negative and positively charged residues, respectively, with a pI value of 4.8 (the C-domain has 27% and 15%, respectively, with a pI value of 4.5). Furthermore, peptide-1 showed an altered electrophoretic mobility (Fig. 4A). The minimal length of the peptide that confers this property is 14 amino acids, as determined by a series of deletion peptides (Table 1). It is well known that pH changes have drastic effects on protein and peptide structures and in particular in peptides that form supramolecular structures. The β-amyloid peptide undergoes conformational change as function of pH. At pH 1.3 and 8.3, adopts a helical conformation, whereas at pH 5.4 it has a β-sheet structure [36]. In the same manner, the ionic self-complementary peptides developed by Zhang and Rich [21,22,37,38] undergo similar structural changes. Peptide-1, when protonated below pH 4, has a CD spectrum characteristic of a canonical β-sheet, similar to that reported for the ionic self-complementary peptide EAK12-d (pI 4.5 – [22]). The structural transition of peptide-1 towards an extended (beta) structure takes place at about pH 5, near its pI value, with an intermediate spectrum (Fig. 4A). Analytical ultracentrifugation demonstrated that in the two pH-dependent conformations peptide-1 forms large oligomers that showed to have amyloid-like properties as is the ability to bind to Congo red and thioflavin T (Fig. 6) and to form fibrils (Fig. 7). The analysis by TEM of the fibrils obtained from freeze-fixed and freeze-dried of peptide-1 solutions at neutral pH showed a regular shape and are long, up to 1000 nm length, and unbranched. However, when the aggregates are prepared from peptide-1 solutions at acidic pH they showed to be more irregular in shape. If the fibrils are composed of the peptide-1 in antiparallel β-sheet conformation, their width should be 6.5 nm in close agreement with the observed average diameter of 8 nm (Fig. 7). Although the detailed structure of fibrils is not available at the moment, a likely model structure of the β-sheet disposition of two peptide-1 molecules was obtained by molecular dynamics. Initially, two peptide-1 molecules in a β-sheet conformation (see Materials and Methods section) were arbitrarily arranged in an antiparallel disposition (here we speculated that in the whole protein the presence of the N-domain will preclude a parallel disposition) with the complementary charged amino acid residues at an intermolecular distance of 4 Å (between the charged atoms – [39]). During the simulation these distances were observed to keep constant, whereas the hydrophobic side chains approached each other. The resulting model is presented in Fig. 8. All nonpolar side chains (blue) project from one face of the β-sheet while all polar side chains (green and magenta) project from the opposing face of the sheet. There was a decrease of the total energy of the system during the simulation, being the main contribution the decrease in the van der Waals energy (data not shown).


Ionic self-complementarity induces amyloid-like fibril formation in an isolated domain of a plant copper metallochaperone protein.

Mira H, Vilar M, Esteve V, Martinell M, Kogan MJ, Giralt E, Salom D, Mingarro I, Peñarrubia L, Pérez-Payá E - BMC Struct. Biol. (2004)

Hypothetical model for peptide-1 fibril formation based on a molecular dynamics simulation. (A) Initial structure with two units of peptide-1 arranged in a β-antiparallel disposition. An intermolecular distance of 4 Å within the complementary charged amino acid residues (between the charged atoms) was used. (B). Snapshot representative of the final part of the simulation where the energy of the system was stabilized. (Blue: hydrophobic residues, green: acidic residues; magenta: basic residues).
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Related In: Results  -  Collection

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Figure 8: Hypothetical model for peptide-1 fibril formation based on a molecular dynamics simulation. (A) Initial structure with two units of peptide-1 arranged in a β-antiparallel disposition. An intermolecular distance of 4 Å within the complementary charged amino acid residues (between the charged atoms) was used. (B). Snapshot representative of the final part of the simulation where the energy of the system was stabilized. (Blue: hydrophobic residues, green: acidic residues; magenta: basic residues).
Mentions: A synthetic peptide (peptide-1 – Fig. 1) derived from the C-domain was selected in order to facilitate the conformational study. Peptide-1 has most of the general sequence properties of the C-domain. It is characterised by a pattern of alternating hydrophobic and hydrophilic residues and by a striking overall charge distribution containing a 30% and 20% of negative and positively charged residues, respectively, with a pI value of 4.8 (the C-domain has 27% and 15%, respectively, with a pI value of 4.5). Furthermore, peptide-1 showed an altered electrophoretic mobility (Fig. 4A). The minimal length of the peptide that confers this property is 14 amino acids, as determined by a series of deletion peptides (Table 1). It is well known that pH changes have drastic effects on protein and peptide structures and in particular in peptides that form supramolecular structures. The β-amyloid peptide undergoes conformational change as function of pH. At pH 1.3 and 8.3, adopts a helical conformation, whereas at pH 5.4 it has a β-sheet structure [36]. In the same manner, the ionic self-complementary peptides developed by Zhang and Rich [21,22,37,38] undergo similar structural changes. Peptide-1, when protonated below pH 4, has a CD spectrum characteristic of a canonical β-sheet, similar to that reported for the ionic self-complementary peptide EAK12-d (pI 4.5 – [22]). The structural transition of peptide-1 towards an extended (beta) structure takes place at about pH 5, near its pI value, with an intermediate spectrum (Fig. 4A). Analytical ultracentrifugation demonstrated that in the two pH-dependent conformations peptide-1 forms large oligomers that showed to have amyloid-like properties as is the ability to bind to Congo red and thioflavin T (Fig. 6) and to form fibrils (Fig. 7). The analysis by TEM of the fibrils obtained from freeze-fixed and freeze-dried of peptide-1 solutions at neutral pH showed a regular shape and are long, up to 1000 nm length, and unbranched. However, when the aggregates are prepared from peptide-1 solutions at acidic pH they showed to be more irregular in shape. If the fibrils are composed of the peptide-1 in antiparallel β-sheet conformation, their width should be 6.5 nm in close agreement with the observed average diameter of 8 nm (Fig. 7). Although the detailed structure of fibrils is not available at the moment, a likely model structure of the β-sheet disposition of two peptide-1 molecules was obtained by molecular dynamics. Initially, two peptide-1 molecules in a β-sheet conformation (see Materials and Methods section) were arbitrarily arranged in an antiparallel disposition (here we speculated that in the whole protein the presence of the N-domain will preclude a parallel disposition) with the complementary charged amino acid residues at an intermolecular distance of 4 Å (between the charged atoms – [39]). During the simulation these distances were observed to keep constant, whereas the hydrophobic side chains approached each other. The resulting model is presented in Fig. 8. All nonpolar side chains (blue) project from one face of the β-sheet while all polar side chains (green and magenta) project from the opposing face of the sheet. There was a decrease of the total energy of the system during the simulation, being the main contribution the decrease in the van der Waals energy (data not shown).

Bottom Line: 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.

View Article: PubMed Central - HTML - PubMed

Affiliation: Departament de Bioquímica i Biologia Molecular, Universitat de València, E-46100 Burjassot, València, Spain. helena.mira@uv.es

ABSTRACT

Background: 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.

Results: 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 beta-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.

Conclusion: 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.

Show MeSH