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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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Conformational characterisation of peptide-1 at 10°C in the presence of TFE and SDS (A). Far-UV CD spectra at pH 4.2 as a function of TFE concentration. TFE induces a decrease in the molar ellipticity at 215 nm (CD spectra acquired at ■, 0 %; ▲, 10 %; Δ, 20 %; ●, 30 %; ○, 40 %, and □ 60 %, v/v, TFE concentrations). (B). Far-UV CD spectra at pH 6.8 at different TFE concentrations (symbols as in panel A). The insert shows the dependence with TFE concentration (v/v) of the molar ellipticity at 215 nm. (C). Far-UV CD spectra at pH 6.8 at different SDS concentrations (■, 0 mM; ◆, 1 mM, ◇, 2 mM; Æ, 3 mM; + 5 mM and ▼, 10 mM). (D). Far-UV CD spectra at pH 3.2 at different SDS concentrations (symbols as in part C). The concentration of peptide-1 in all the spectra was 50 μM.
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Figure 5: Conformational characterisation of peptide-1 at 10°C in the presence of TFE and SDS (A). Far-UV CD spectra at pH 4.2 as a function of TFE concentration. TFE induces a decrease in the molar ellipticity at 215 nm (CD spectra acquired at ■, 0 %; ▲, 10 %; Δ, 20 %; ●, 30 %; ○, 40 %, and □ 60 %, v/v, TFE concentrations). (B). Far-UV CD spectra at pH 6.8 at different TFE concentrations (symbols as in panel A). The insert shows the dependence with TFE concentration (v/v) of the molar ellipticity at 215 nm. (C). Far-UV CD spectra at pH 6.8 at different SDS concentrations (■, 0 mM; ◆, 1 mM, ◇, 2 mM; Æ, 3 mM; + 5 mM and ▼, 10 mM). (D). Far-UV CD spectra at pH 3.2 at different SDS concentrations (symbols as in part C). The concentration of peptide-1 in all the spectra was 50 μM.

Mentions: TFE titration of a peptide-1 solution at pH 4.2 induced the stabilization of the β-sheet conformation (Fig. 5A). The CD spectra corresponding to the peptide-1 either in the absence or in the presence of up to 20% TFE (v/v) showed an isodichroic point at 203 nm that suggested a single conformational transition. However, at TFE concentrations of 40 and 60% (v/v), the spectral characteristics denoted the presence of a mixture of α-helical and β-sheet conformations and the isodichroic point shifted to 201 nm. TFE titration of a solution of peptide-1 in extended (beta) conformation at pH 6.8 induced the transition towards a canonical β-sheet conformation (Fig. 5B). Thus, at low TFE concentrations (below 20%, v/v) the CD spectra showed a negative ellipticity band at 198 nm, while when the TFE concentration was increased, the CD spectra displayed a single minimum at 215 nm characteristic of β-sheet structure (Fig. 5B and insert). The conformational transition of peptide-1 was also analysed in the presence of SDS, which is considered a template that would stabilize both α-helical and β-sheet conformations depending on the intrinsic propensity of the polypeptide sequence to adopt a preferred secondary structure [31]. At neutral pH concentrations of SDS below 2 mM induced the stabilization of the extended (beta) structure in peptide-1, whereas higher SDS concentrations induced an α-helical conformation (Fig. 5C). At acidic pH values, it has been shown that peptide-1 adopts a β-sheet conformation that renders CD spectra characterized by a minimum at 215 nm (Fig. 4B). When the influence of SDS on the conformational transition of peptide-1 was analyzed at pH 3.2 a further increase in the characteristics that define the canonical CD spectra associated to the β-sheet conformation was obtained. In this sense, both the ellipticity at 215 nm (in absolute value) and at 198 nm increased (Fig. 5D).


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)

Conformational characterisation of peptide-1 at 10°C in the presence of TFE and SDS (A). Far-UV CD spectra at pH 4.2 as a function of TFE concentration. TFE induces a decrease in the molar ellipticity at 215 nm (CD spectra acquired at ■, 0 %; ▲, 10 %; Δ, 20 %; ●, 30 %; ○, 40 %, and □ 60 %, v/v, TFE concentrations). (B). Far-UV CD spectra at pH 6.8 at different TFE concentrations (symbols as in panel A). The insert shows the dependence with TFE concentration (v/v) of the molar ellipticity at 215 nm. (C). Far-UV CD spectra at pH 6.8 at different SDS concentrations (■, 0 mM; ◆, 1 mM, ◇, 2 mM; Æ, 3 mM; + 5 mM and ▼, 10 mM). (D). Far-UV CD spectra at pH 3.2 at different SDS concentrations (symbols as in part C). The concentration of peptide-1 in all the spectra was 50 μM.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC425589&req=5

Figure 5: Conformational characterisation of peptide-1 at 10°C in the presence of TFE and SDS (A). Far-UV CD spectra at pH 4.2 as a function of TFE concentration. TFE induces a decrease in the molar ellipticity at 215 nm (CD spectra acquired at ■, 0 %; ▲, 10 %; Δ, 20 %; ●, 30 %; ○, 40 %, and □ 60 %, v/v, TFE concentrations). (B). Far-UV CD spectra at pH 6.8 at different TFE concentrations (symbols as in panel A). The insert shows the dependence with TFE concentration (v/v) of the molar ellipticity at 215 nm. (C). Far-UV CD spectra at pH 6.8 at different SDS concentrations (■, 0 mM; ◆, 1 mM, ◇, 2 mM; Æ, 3 mM; + 5 mM and ▼, 10 mM). (D). Far-UV CD spectra at pH 3.2 at different SDS concentrations (symbols as in part C). The concentration of peptide-1 in all the spectra was 50 μM.
Mentions: TFE titration of a peptide-1 solution at pH 4.2 induced the stabilization of the β-sheet conformation (Fig. 5A). The CD spectra corresponding to the peptide-1 either in the absence or in the presence of up to 20% TFE (v/v) showed an isodichroic point at 203 nm that suggested a single conformational transition. However, at TFE concentrations of 40 and 60% (v/v), the spectral characteristics denoted the presence of a mixture of α-helical and β-sheet conformations and the isodichroic point shifted to 201 nm. TFE titration of a solution of peptide-1 in extended (beta) conformation at pH 6.8 induced the transition towards a canonical β-sheet conformation (Fig. 5B). Thus, at low TFE concentrations (below 20%, v/v) the CD spectra showed a negative ellipticity band at 198 nm, while when the TFE concentration was increased, the CD spectra displayed a single minimum at 215 nm characteristic of β-sheet structure (Fig. 5B and insert). The conformational transition of peptide-1 was also analysed in the presence of SDS, which is considered a template that would stabilize both α-helical and β-sheet conformations depending on the intrinsic propensity of the polypeptide sequence to adopt a preferred secondary structure [31]. At neutral pH concentrations of SDS below 2 mM induced the stabilization of the extended (beta) structure in peptide-1, whereas higher SDS concentrations induced an α-helical conformation (Fig. 5C). At acidic pH values, it has been shown that peptide-1 adopts a β-sheet conformation that renders CD spectra characterized by a minimum at 215 nm (Fig. 4B). When the influence of SDS on the conformational transition of peptide-1 was analyzed at pH 3.2 a further increase in the characteristics that define the canonical CD spectra associated to the β-sheet conformation was obtained. In this sense, both the ellipticity at 215 nm (in absolute value) and at 198 nm increased (Fig. 5D).

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
Related in: MedlinePlus