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PrP charge structure encodes interdomain interactions.

Martínez J, Sánchez R, Castellanos M, Makarava N, Aguzzi A, Baskakov IV, Gasset M - Sci Rep (2015)

Bottom Line: We found that charges contain the information for interdomain interactions.Independently of this structural effect, the N-terminal electropositive clusters regulate the α-cleavage efficiency.These findings show that the PrP charge structure functions as a code set up to ensure function and reduce pathogenic routes.

View Article: PubMed Central - PubMed

Affiliation: Instituto Química-Física "Rocasolano", Consejo Superior de Investigaciones Científicas, Madrid 28006, Spain.

ABSTRACT
Almost all proteins contain charged residues, and their chain distribution is tailored to fulfill essential ionic interactions for folding, binding and catalysis. Among proteins, the hinged two-domain chain of the cellular prion protein (PrP(C)) exhibits a peculiar charge structure with unclear consequences in its structural malleability. To decipher the charge design role, we generated charge-reverted mutants for each domain and analyzed their effect on conformational and metabolic features. We found that charges contain the information for interdomain interactions. Use of dynamic light scattering and thermal denaturation experiments delineates the compaction of the α-fold by an electrostatic compensation between the polybasic 23-30 region and the α3 electronegative surface. This interaction increases stability and disfavors fibrillation. Independently of this structural effect, the N-terminal electropositive clusters regulate the α-cleavage efficiency. In the fibrillar state, use of circular dichroism, atomic-force and fluorescence microscopies reveal that the N-terminal positive clusters and the α3 electronegative surface dictate the secondary structure, the assembly hierarchy and the growth length of the fibril state. These findings show that the PrP charge structure functions as a code set up to ensure function and reduce pathogenic routes.

No MeSH data available.


Related in: MedlinePlus

Charge changes modify the fibrillation propensity and processing of PrP.Time-dependence of ThT binding of the PrP wt and mutants (40 μM protein concentrations) in 50 mM MES pH 6.5 at 37 °C containing (a) 2 M GdnCl and (b) 3 M urea and 50 mM NaCl. The curves represent the average of three independent measurements, performed in triplicate. (c) Lag-phases of the fibrillation reactions of the PrP wt and mutants in 50 mM Mes pH 6.5 containing either 2 M GdnCl or 3 M urea with 50 mM NaCl. The depicted values correspond to three independent experiments, each performed in triplicate. (d) Western blot of PNGase-treated cell lysates of CHO cells transfected with PrP wt and the charged mutants. Detection was performed using POM17, and the positions of the full-length (FL) and N-terminal-truncated (C1 fragment) chains are depicted. (e) Variations in the C1/FL ratio of the PrP chains. Quantifications are the average of two independent transfection assays. Error bar represents the standard deviation (s.d.). *p < 0.01.
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f4: Charge changes modify the fibrillation propensity and processing of PrP.Time-dependence of ThT binding of the PrP wt and mutants (40 μM protein concentrations) in 50 mM MES pH 6.5 at 37 °C containing (a) 2 M GdnCl and (b) 3 M urea and 50 mM NaCl. The curves represent the average of three independent measurements, performed in triplicate. (c) Lag-phases of the fibrillation reactions of the PrP wt and mutants in 50 mM Mes pH 6.5 containing either 2 M GdnCl or 3 M urea with 50 mM NaCl. The depicted values correspond to three independent experiments, each performed in triplicate. (d) Western blot of PNGase-treated cell lysates of CHO cells transfected with PrP wt and the charged mutants. Detection was performed using POM17, and the positions of the full-length (FL) and N-terminal-truncated (C1 fragment) chains are depicted. (e) Variations in the C1/FL ratio of the PrP chains. Quantifications are the average of two independent transfection assays. Error bar represents the standard deviation (s.d.). *p < 0.01.

Mentions: Although most PrP amyloids generated in vitro lack the infectivity and proteolytic signatures of PrPSc, their formation models the chain propensity and the conformational changes required for GD self-assembly5556. To test whether the charge structure, through either the α-fold stabilization described above or the initial self-assembly step, impacts the fibrillation, we performed time-dependent Thioflavin T (ThT) binding experiments using the various PrP chains at pH 6.5 and calculated the lag-phase as indicator of propensity (Fig. 4). To induce the required mild denaturation, we used either 2 M GdnCl (conventional ionic media) or 3 M urea containing 50 mM NaCl (low salt). As anticipated from the exposed character of the mutated charges and the high ionic strength of the media, fibrillation in 2 M GdnCl (Fig. 4a) yielded lag-phases that were roughly similar for all chains, with minor reductions (K2,K4) or enhancements (K6) (Fig. 4c). On the contrary, reactions in the low salt media containing 3 M urea and 50 mM NaCl revealed that with the exception of Q219K, all mutants form fibrils faster than the PrP wt as indicated by their higher lag-phases (Fig. 4b,c). Kinetic curves also showed that charge changes in the GD (E200K, Q217R, Q219K and E221K) decrease the ThT fluorescence increment linked to fibrillation predominantly under low salt (Fig. 4a,b), suggesting off-pathway events.


PrP charge structure encodes interdomain interactions.

Martínez J, Sánchez R, Castellanos M, Makarava N, Aguzzi A, Baskakov IV, Gasset M - Sci Rep (2015)

Charge changes modify the fibrillation propensity and processing of PrP.Time-dependence of ThT binding of the PrP wt and mutants (40 μM protein concentrations) in 50 mM MES pH 6.5 at 37 °C containing (a) 2 M GdnCl and (b) 3 M urea and 50 mM NaCl. The curves represent the average of three independent measurements, performed in triplicate. (c) Lag-phases of the fibrillation reactions of the PrP wt and mutants in 50 mM Mes pH 6.5 containing either 2 M GdnCl or 3 M urea with 50 mM NaCl. The depicted values correspond to three independent experiments, each performed in triplicate. (d) Western blot of PNGase-treated cell lysates of CHO cells transfected with PrP wt and the charged mutants. Detection was performed using POM17, and the positions of the full-length (FL) and N-terminal-truncated (C1 fragment) chains are depicted. (e) Variations in the C1/FL ratio of the PrP chains. Quantifications are the average of two independent transfection assays. Error bar represents the standard deviation (s.d.). *p < 0.01.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
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f4: Charge changes modify the fibrillation propensity and processing of PrP.Time-dependence of ThT binding of the PrP wt and mutants (40 μM protein concentrations) in 50 mM MES pH 6.5 at 37 °C containing (a) 2 M GdnCl and (b) 3 M urea and 50 mM NaCl. The curves represent the average of three independent measurements, performed in triplicate. (c) Lag-phases of the fibrillation reactions of the PrP wt and mutants in 50 mM Mes pH 6.5 containing either 2 M GdnCl or 3 M urea with 50 mM NaCl. The depicted values correspond to three independent experiments, each performed in triplicate. (d) Western blot of PNGase-treated cell lysates of CHO cells transfected with PrP wt and the charged mutants. Detection was performed using POM17, and the positions of the full-length (FL) and N-terminal-truncated (C1 fragment) chains are depicted. (e) Variations in the C1/FL ratio of the PrP chains. Quantifications are the average of two independent transfection assays. Error bar represents the standard deviation (s.d.). *p < 0.01.
Mentions: Although most PrP amyloids generated in vitro lack the infectivity and proteolytic signatures of PrPSc, their formation models the chain propensity and the conformational changes required for GD self-assembly5556. To test whether the charge structure, through either the α-fold stabilization described above or the initial self-assembly step, impacts the fibrillation, we performed time-dependent Thioflavin T (ThT) binding experiments using the various PrP chains at pH 6.5 and calculated the lag-phase as indicator of propensity (Fig. 4). To induce the required mild denaturation, we used either 2 M GdnCl (conventional ionic media) or 3 M urea containing 50 mM NaCl (low salt). As anticipated from the exposed character of the mutated charges and the high ionic strength of the media, fibrillation in 2 M GdnCl (Fig. 4a) yielded lag-phases that were roughly similar for all chains, with minor reductions (K2,K4) or enhancements (K6) (Fig. 4c). On the contrary, reactions in the low salt media containing 3 M urea and 50 mM NaCl revealed that with the exception of Q219K, all mutants form fibrils faster than the PrP wt as indicated by their higher lag-phases (Fig. 4b,c). Kinetic curves also showed that charge changes in the GD (E200K, Q217R, Q219K and E221K) decrease the ThT fluorescence increment linked to fibrillation predominantly under low salt (Fig. 4a,b), suggesting off-pathway events.

Bottom Line: We found that charges contain the information for interdomain interactions.Independently of this structural effect, the N-terminal electropositive clusters regulate the α-cleavage efficiency.These findings show that the PrP charge structure functions as a code set up to ensure function and reduce pathogenic routes.

View Article: PubMed Central - PubMed

Affiliation: Instituto Química-Física "Rocasolano", Consejo Superior de Investigaciones Científicas, Madrid 28006, Spain.

ABSTRACT
Almost all proteins contain charged residues, and their chain distribution is tailored to fulfill essential ionic interactions for folding, binding and catalysis. Among proteins, the hinged two-domain chain of the cellular prion protein (PrP(C)) exhibits a peculiar charge structure with unclear consequences in its structural malleability. To decipher the charge design role, we generated charge-reverted mutants for each domain and analyzed their effect on conformational and metabolic features. We found that charges contain the information for interdomain interactions. Use of dynamic light scattering and thermal denaturation experiments delineates the compaction of the α-fold by an electrostatic compensation between the polybasic 23-30 region and the α3 electronegative surface. This interaction increases stability and disfavors fibrillation. Independently of this structural effect, the N-terminal electropositive clusters regulate the α-cleavage efficiency. In the fibrillar state, use of circular dichroism, atomic-force and fluorescence microscopies reveal that the N-terminal positive clusters and the α3 electronegative surface dictate the secondary structure, the assembly hierarchy and the growth length of the fibril state. These findings show that the PrP charge structure functions as a code set up to ensure function and reduce pathogenic routes.

No MeSH data available.


Related in: MedlinePlus