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Calcium binding promotes prion protein fragment 90-231 conformational change toward a membrane destabilizing and cytotoxic structure.

ABSTRACT: The pathological form of prion protein (PrP(Sc)), as other amyloidogenic proteins, causes a marked increase of membrane permeability. PrP(Sc) extracted from infected Syrian hamster brains induces a considerable change in membrane ionic conductance, although the contribution of this interaction to the molecular mechanism of neurodegeneration process is still controversial. We previously showed that the human PrP fragment 90-231 (hPrP??????) increases ionic conductance across artificial lipid bilayer, in a calcium-dependent manner, producing an alteration similar to that observed for PrP(Sc). In the present study we demonstrate that hPrP??????, pre-incubated with 10 mM Ca?? and then re-suspended in physiological external solution increases not only membrane conductance but neurotoxicity as well. Furthermore we show the existence of a direct link between these two effects as demonstrated by a highly statistically significant correlation in several experimental conditions. A similar correlation between increased membrane conductance and cell degeneration has been observed assaying hPrP?????? bearing pathogenic mutations (D202N and E200K). We also report that Ca?? binding to hPrP?????? induces a conformational change based on an alteration of secondary structure characterized by loss of alpha-helix content causing hydrophobic amino acid exposure and proteinase K resistance. These features, either acquired after controlled thermal denaturation or induced by D202N and E200K mutations were previously identified as responsible for hPrP?????? cytotoxicity. Finally, by in silico structural analysis, we propose that Ca?? binding to hPrP?????? modifies amino acid orientation, in the same way induced by E200K mutation, thus suggesting a pathway for the structural alterations responsible of PrP neurotoxicity.

SUBMITTER: Sorrentino S

PROVIDER: S-EPMC3394757 | biostudies-literature | 2012

REPOSITORIES: biostudies-literature

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Calcium binding promotes prion protein fragment 90-231 conformational change toward a membrane destabilizing and cytotoxic structure.

Sorrentino Sacha S Bucciarelli Tonino T Corsaro Alessandro A Tosatto Alessio A Thellung Stefano S Villa Valentina V Schininà M Eugenia ME Maras Bruno B Galeno Roberta R Scotti Luca L Creati Francesco F Marrone Alessandro A Re Nazzareno N Aceto Antonio A Florio Tullio T Mazzanti Michele M

PloS one 20120711 7

The pathological form of prion protein (PrP(Sc)), as other amyloidogenic proteins, causes a marked increase of membrane permeability. PrP(Sc) extracted from infected Syrian hamster brains induces a considerable change in membrane ionic conductance, although the contribution of this interaction to the molecular mechanism of neurodegeneration process is still controversial. We previously showed that the human PrP fragment 90-231 (hPrP₉₀₋₂₃₁) increases ionic conductance across artificial lipid bila ...[more]

PMID: 22811758

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Project description:BackgroundRicin is a potent toxin and known bioterrorism threat with no available antidote. The ricin A-chain (RTA) acts enzymatically to cleave a specific adenine base from ribosomal RNA, thereby blocking translation. To understand better the relationship between ligand binding and RTA active site conformational change, we used a fragment-based approach to find a minimal set of bonding interactions able to induce rearrangements in critical side-chain positions.ResultsWe found that the smallest ligand stabilizing an open conformer of the RTA active site pocket was an amide group, bound weakly by only a few hydrogen bonds to the protein. Complexes with small amide-containing molecules also revealed a switch in geometry from a parallel towards a splayed arrangement of an arginine-tryptophan cation-pi interaction that was associated with an increase and red-shift in tryptophan fluorescence upon ligand binding. Using the observed fluorescence signal, we determined the thermodynamic changes of adenine binding to the RTA active site, as well as the site-specific binding of urea. Urea binding had a favorable enthalpy change and unfavorable entropy change, with a DeltaH of -13 +/- 2 kJ/mol and a DeltaS of -0.04 +/- 0.01 kJ/(K*mol). The side-chain position of residue Tyr80 in a complex with adenine was found not to involve as large an overlap of rings with the purine as previously considered, suggesting a smaller role for aromatic stacking at the RTA active site.ConclusionWe found that amide ligands can bind weakly but specifically to the ricin active site, producing significant shifts in positions of the critical active site residues Arg180 and Tyr80. These results indicate that fragment-based drug discovery methods are capable of identifying minimal bonding determinants of active-site side-chain rearrangements and the mechanistic origins of spectroscopic shifts. Our results suggest that tryptophan fluorescence provides a sensitive probe for the geometric relationship of arginine-tryptophan pairs, which often have significant roles in protein function. Using the unusual characteristics of the RTA system, we measured the still controversial thermodynamic changes of site-specific urea binding to a protein, results that are relevant to understanding the physical mechanisms of protein denaturation.

Dataset Information

Calcium binding promotes prion protein fragment 90-231 conformational change toward a membrane destabilizing and cytotoxic structure.

Publications

Calcium binding promotes prion protein fragment 90-231 conformational change toward a membrane destabilizing and cytotoxic structure.

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