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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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Representation of the C-domain of the CCH protein and related synthetic peptides. (A). Amino acid sequence (one letter code) of the C-domain (residues 69–121) of CCH. The sequence shows relative alternating periodicity of nonpolar (●) and polar (○) amino acids. The stretch of 'h' indicates the putative α-helix predicted by secondary-structure algorithms. The bars denote the location of the two 16-residue repeats of binary structural periodicity of polar/non polar amino acids. (B). Such a motif was used to design the synthetic peptide-1. The amino acid sequence of peptide-1 is arranged into an α-helix (C) and into a β-sheet conformation (D). Positively and negatively charged amino acids are in blue and red, respectively and hydrophobic amino acids are bold cycled.
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Figure 1: Representation of the C-domain of the CCH protein and related synthetic peptides. (A). Amino acid sequence (one letter code) of the C-domain (residues 69–121) of CCH. The sequence shows relative alternating periodicity of nonpolar (●) and polar (○) amino acids. The stretch of 'h' indicates the putative α-helix predicted by secondary-structure algorithms. The bars denote the location of the two 16-residue repeats of binary structural periodicity of polar/non polar amino acids. (B). Such a motif was used to design the synthetic peptide-1. The amino acid sequence of peptide-1 is arranged into an α-helix (C) and into a β-sheet conformation (D). Positively and negatively charged amino acids are in blue and red, respectively and hydrophobic amino acids are bold cycled.

Mentions: The present study describes the amyloidosis properties of the C-terminal domain of the Arabidopsis thaliana copper metallochaperone CCH, which is a functional homologue of the cytosolic metallochaperone antioxidant 1 (Atx1) [10] from the yeast Saccharomyces cerevisiae. Atx1 delivers copper to transporters at a post-Golgi compartment to load the ion at cuproproteins in the secretory pathway [11,12]. CCH has been found in plant vascular tissues where its concentration increases greatly during senescence [13] and it is mainly located in cells that lack their nuclei and therefore they could be unable of DNA transcription or translation. Consequently, CCH should be transported from neighbour cells through plasmodesmata, being the only metallochaperone reported to date that could leave the cell where it is synthesised [13]. CCH (121 residues) has two different domains with independent folding pathways [14]. The conserved N-terminal domain (amino acids 1–68, referred herein as the N-domain) contains the copper-binding motif MXCXXC that retains both, the overall "open-faced β-sandwich" fold and the copper chaperone and antioxidant properties described for the yeast Atx1 [14]. The C-terminal domain (amino acids 69–121, the C-domain), is absent in all the identified non-plant metallochaperones [10], displayed altered SDS/PAGE mobility and it adopts an extended (beta) conformation in solution and in the presence of anionic detergents. Thus, we have postulated that the CCH extra C-domain participates in this plant-exclusive copper transport mechanism. The C-domain has structural features that could favour the adoption of an extended structure in β-sheet conformation. It is characterized by two repeats (Fig. 1) that have a binary structural periodicity of polar/non polar amino acids along the sequence with potential to adopt β-sheet structures that self-assembled into large oligomers [15,16]. In this report we have addressed the structural characterization of the C-domain. The conformational behaviour of the molecule in solution is complex and implies the adoption of mixed conformations in different environments. In an increasing number of proteins, the understanding of the folding mechanism could be addressed by means of the analysis of short polypeptide segments [17-20]. Thus a fruitful approach to investigate the autonomous folding and function of protein subdomains is the design and structural characterization of simplified peptide models that could reproduce the molecular characteristics of the whole protein domain. In the present study, we have selected the peptide, K90TEAETKTEAKVDAKADVE108 derived from the C-domain of CCH, since it contains a segment of alternating hydrophobic and hydrophilic residues and a striking overall charge distribution (Fig. 1). The peptide adopts an 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. The results showed that polypeptides containing complementary charges promote fibril formation, not only in de novo designed peptides [21,22] but also in polypeptides derived from natural existing proteins. Furthermore, the relevance of the self-assembly properties of the C-domain of CCH is discussed in the context of plasmodesmata trafficking through microtubules and other putative functions related to the role of its ortologues.


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)

Representation of the C-domain of the CCH protein and related synthetic peptides. (A). Amino acid sequence (one letter code) of the C-domain (residues 69–121) of CCH. The sequence shows relative alternating periodicity of nonpolar (●) and polar (○) amino acids. The stretch of 'h' indicates the putative α-helix predicted by secondary-structure algorithms. The bars denote the location of the two 16-residue repeats of binary structural periodicity of polar/non polar amino acids. (B). Such a motif was used to design the synthetic peptide-1. The amino acid sequence of peptide-1 is arranged into an α-helix (C) and into a β-sheet conformation (D). Positively and negatively charged amino acids are in blue and red, respectively and hydrophobic amino acids are bold cycled.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC425589&req=5

Figure 1: Representation of the C-domain of the CCH protein and related synthetic peptides. (A). Amino acid sequence (one letter code) of the C-domain (residues 69–121) of CCH. The sequence shows relative alternating periodicity of nonpolar (●) and polar (○) amino acids. The stretch of 'h' indicates the putative α-helix predicted by secondary-structure algorithms. The bars denote the location of the two 16-residue repeats of binary structural periodicity of polar/non polar amino acids. (B). Such a motif was used to design the synthetic peptide-1. The amino acid sequence of peptide-1 is arranged into an α-helix (C) and into a β-sheet conformation (D). Positively and negatively charged amino acids are in blue and red, respectively and hydrophobic amino acids are bold cycled.
Mentions: The present study describes the amyloidosis properties of the C-terminal domain of the Arabidopsis thaliana copper metallochaperone CCH, which is a functional homologue of the cytosolic metallochaperone antioxidant 1 (Atx1) [10] from the yeast Saccharomyces cerevisiae. Atx1 delivers copper to transporters at a post-Golgi compartment to load the ion at cuproproteins in the secretory pathway [11,12]. CCH has been found in plant vascular tissues where its concentration increases greatly during senescence [13] and it is mainly located in cells that lack their nuclei and therefore they could be unable of DNA transcription or translation. Consequently, CCH should be transported from neighbour cells through plasmodesmata, being the only metallochaperone reported to date that could leave the cell where it is synthesised [13]. CCH (121 residues) has two different domains with independent folding pathways [14]. The conserved N-terminal domain (amino acids 1–68, referred herein as the N-domain) contains the copper-binding motif MXCXXC that retains both, the overall "open-faced β-sandwich" fold and the copper chaperone and antioxidant properties described for the yeast Atx1 [14]. The C-terminal domain (amino acids 69–121, the C-domain), is absent in all the identified non-plant metallochaperones [10], displayed altered SDS/PAGE mobility and it adopts an extended (beta) conformation in solution and in the presence of anionic detergents. Thus, we have postulated that the CCH extra C-domain participates in this plant-exclusive copper transport mechanism. The C-domain has structural features that could favour the adoption of an extended structure in β-sheet conformation. It is characterized by two repeats (Fig. 1) that have a binary structural periodicity of polar/non polar amino acids along the sequence with potential to adopt β-sheet structures that self-assembled into large oligomers [15,16]. In this report we have addressed the structural characterization of the C-domain. The conformational behaviour of the molecule in solution is complex and implies the adoption of mixed conformations in different environments. In an increasing number of proteins, the understanding of the folding mechanism could be addressed by means of the analysis of short polypeptide segments [17-20]. Thus a fruitful approach to investigate the autonomous folding and function of protein subdomains is the design and structural characterization of simplified peptide models that could reproduce the molecular characteristics of the whole protein domain. In the present study, we have selected the peptide, K90TEAETKTEAKVDAKADVE108 derived from the C-domain of CCH, since it contains a segment of alternating hydrophobic and hydrophilic residues and a striking overall charge distribution (Fig. 1). The peptide adopts an 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. The results showed that polypeptides containing complementary charges promote fibril formation, not only in de novo designed peptides [21,22] but also in polypeptides derived from natural existing proteins. Furthermore, the relevance of the self-assembly properties of the C-domain of CCH is discussed in the context of plasmodesmata trafficking through microtubules and other putative functions related to the role of its ortologues.

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