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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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TEM images. TEM images of the replicas obtained after freeze-fixation and freeze-drying of an aqueous solution of recombinant C-domain at different pHs. (Bar = 0.2 μm).
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Figure 2: TEM images. TEM images of the replicas obtained after freeze-fixation and freeze-drying of an aqueous solution of recombinant C-domain at different pHs. (Bar = 0.2 μm).

Mentions: The recombinant C-domain of CCH, expressed and purified from Escherichia coli, has been shown to have an altered electrophoretic mobility in SDS-PAGE and its preliminary structural characterization suggested that it could adopt an extended β-sheet structure in solution at neutral pH [14]. The amino acid sequence of the C-domain is characterized by the presence of two segments of binary structural periodicity of polar/non polar amino acids (Fig. 1A). Provided that 22 out of 30 polar residues are Asp, Glu and Lys, the conformational behaviour of the C-domain could be influenced by ionic self-complementarity that has been early described as a property that confers multifaceted behaviour in model peptides and that could induce fibril formation [22]. In fact the polypeptide that defines the C-domain showed a complex behaviour in solution with the coexistence of monomeric and large oligomeric conformations. The presence in salt-free aqueous solution of large fibril-like aggregates was demonstrated by freeze fixation, freeze drying transmission electron microscopy imaging (TEM) (Fig. 2). A recombinant C-domain polypeptide solution was prepared and divided in four fractions adjusted with HCl or NaOH at different pH values. At pH 7 and 9 the fibrils are long and unbranched while at pH 3 and 5 they showed irregular shapes. As previously mentioned, the recombinant CCH C-domain has been shown to present an altered mobility in SDS-PAGE gels [14]. Electrophoretic mobilities on SDS-PAGE uncorrelated to protein molecular masses, have been described as a result of intrinsic net charge, SDS-induced protein conformation and/or SDS-resistant oligomerization [23-26]. We first examined the aggregation state of recombinant CCH C-domain by sedimentation equilibrium at three different protein concentrations in 50 mM Tris-HCl buffer pH 8.0 containing 0.2 M NaCl that precludes the oligomerization of the polypeptide induced by ionic self-complementarity (Fig. 3A). Three data sets were fit simultaneously to a single species model, with molecular weight as a global fitting parameter. The experimental molecular weight obtained was 4910 ± 140 Da, which fits well to the expected value of 4936.5 Da derived from the amino acid sequence of the C-domain. However, these results differ from the molecular weight obtained in SDS-PAGE for the recombinant C-domain [14]. Since SDS has been described as a membrane mimetic agent prone to induce peptide conformational changes [27] that in turn can modify protein-protein interactions, it is possible that SDS could orchestrate conformational changes leading to oligomerization. Therefore, we performed analytical ultracentrifugation in the presence of SDS at submicellar concentrations (1 mM), like in the SDS-PAGE experiment. The sedimentation profile of the recombinant C-domain in these conditions (black squares in Fig 3B) did not fit well to a model representing a single species model (not shown). Then we fit the data to a two state equilibrium model (i.e., monomer ↔ n-mer) where the aggregation number (n) and the equilibrium dissociation constant are the global fitting parameters, and the monomer molecular weight (4936.5 Da) is held constant (solid line in Fig 3B). The result obtained was n = 7.3 ± 0.2, and the equilibrium dissociation constant value suggested the presence of significant amounts of both, monomers and oligomers in the sample. This model, however, must be considered only as an approximation to a more realistic model in which the monomers and oligomers would associate with SDS molecules. No further attempt was made to fit the data to a more complex equilibrium model due to the uncertainty in the stoichiometry of the protein-SDS interaction, and the fact that the C-domain and SDS have different partial specific volumes (0.7404 mL/mg calculated for the C-domain, and 0.859 mL/mg for SDS below critical micelle concentration – [28]). From these results and those obtained in TEM, SDS-PAGE and CD, it could be concluded that the C-domain of CCH protein has tendency to form oligomers in different experimental conditions.


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)

TEM images. TEM images of the replicas obtained after freeze-fixation and freeze-drying of an aqueous solution of recombinant C-domain at different pHs. (Bar = 0.2 μm).
© Copyright Policy
Related In: Results  -  Collection

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

Figure 2: TEM images. TEM images of the replicas obtained after freeze-fixation and freeze-drying of an aqueous solution of recombinant C-domain at different pHs. (Bar = 0.2 μm).
Mentions: The recombinant C-domain of CCH, expressed and purified from Escherichia coli, has been shown to have an altered electrophoretic mobility in SDS-PAGE and its preliminary structural characterization suggested that it could adopt an extended β-sheet structure in solution at neutral pH [14]. The amino acid sequence of the C-domain is characterized by the presence of two segments of binary structural periodicity of polar/non polar amino acids (Fig. 1A). Provided that 22 out of 30 polar residues are Asp, Glu and Lys, the conformational behaviour of the C-domain could be influenced by ionic self-complementarity that has been early described as a property that confers multifaceted behaviour in model peptides and that could induce fibril formation [22]. In fact the polypeptide that defines the C-domain showed a complex behaviour in solution with the coexistence of monomeric and large oligomeric conformations. The presence in salt-free aqueous solution of large fibril-like aggregates was demonstrated by freeze fixation, freeze drying transmission electron microscopy imaging (TEM) (Fig. 2). A recombinant C-domain polypeptide solution was prepared and divided in four fractions adjusted with HCl or NaOH at different pH values. At pH 7 and 9 the fibrils are long and unbranched while at pH 3 and 5 they showed irregular shapes. As previously mentioned, the recombinant CCH C-domain has been shown to present an altered mobility in SDS-PAGE gels [14]. Electrophoretic mobilities on SDS-PAGE uncorrelated to protein molecular masses, have been described as a result of intrinsic net charge, SDS-induced protein conformation and/or SDS-resistant oligomerization [23-26]. We first examined the aggregation state of recombinant CCH C-domain by sedimentation equilibrium at three different protein concentrations in 50 mM Tris-HCl buffer pH 8.0 containing 0.2 M NaCl that precludes the oligomerization of the polypeptide induced by ionic self-complementarity (Fig. 3A). Three data sets were fit simultaneously to a single species model, with molecular weight as a global fitting parameter. The experimental molecular weight obtained was 4910 ± 140 Da, which fits well to the expected value of 4936.5 Da derived from the amino acid sequence of the C-domain. However, these results differ from the molecular weight obtained in SDS-PAGE for the recombinant C-domain [14]. Since SDS has been described as a membrane mimetic agent prone to induce peptide conformational changes [27] that in turn can modify protein-protein interactions, it is possible that SDS could orchestrate conformational changes leading to oligomerization. Therefore, we performed analytical ultracentrifugation in the presence of SDS at submicellar concentrations (1 mM), like in the SDS-PAGE experiment. The sedimentation profile of the recombinant C-domain in these conditions (black squares in Fig 3B) did not fit well to a model representing a single species model (not shown). Then we fit the data to a two state equilibrium model (i.e., monomer ↔ n-mer) where the aggregation number (n) and the equilibrium dissociation constant are the global fitting parameters, and the monomer molecular weight (4936.5 Da) is held constant (solid line in Fig 3B). The result obtained was n = 7.3 ± 0.2, and the equilibrium dissociation constant value suggested the presence of significant amounts of both, monomers and oligomers in the sample. This model, however, must be considered only as an approximation to a more realistic model in which the monomers and oligomers would associate with SDS molecules. No further attempt was made to fit the data to a more complex equilibrium model due to the uncertainty in the stoichiometry of the protein-SDS interaction, and the fact that the C-domain and SDS have different partial specific volumes (0.7404 mL/mg calculated for the C-domain, and 0.859 mL/mg for SDS below critical micelle concentration – [28]). From these results and those obtained in TEM, SDS-PAGE and CD, it could be concluded that the C-domain of CCH protein has tendency to form oligomers in different experimental conditions.

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