Membrane Interactions and Conformational Preferences of Human and Avian Prion N-Terminal Tandem Repeats: The Role of Copper(II) Ions, pH, and Membrane Mimicking Environments

Abstract

The flexible N-terminal domain of the prion protein (PrP(c)) is believed to play a pivotal role in both trafficking of the protein through the cell membrane and its pathogenic conversion into the β sheet-rich scrapie isoform (PrP(sc)). Unlike mammalian PrP(c), avian prion proteins are not known to undergo any pathogenic conformational conversions. Consequently, some critical advances in our understanding of the molecular mechanisms underlying prion pathogenesis are expected from comparative studies of the biophysical properties of the N-terminal domains of the two proteins. The present study addresses the role played by different environmental factors, i.e., copper(II), pH, and membrane-mimicking environments, in assisting the conformational preferences of huPrP60-91 and chPrP53-76, two soluble peptides encompassing the N-terminal copper(II) binding domains of the human and chicken prion proteins, respectively. Moreover, the membrane interactions of huPrP60-91, chPrP53-76, and their copper(II) complexes were evaluated by Trp fluorescence in conjunction with measurements of the variation in thermotropic properties of 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) unilamellar vesicles. Circular dichroism experiments revealed that huPrP60-91 adopts a predominant polyproline II conformation in aqueous solution that is destabilized at basic pH or in the presence of trifluoroethanol (TFE). Unlike anionic sodium dodecyl sulfate (SDS), which seems to stabilize the polyproline II conformation further, zwitterionic dodecylphosphocholine (DPC) micelles do not affect the peptide structure. On the contrary, copper(II) promptly promotes an increase in β-turn-rich structures. Differential scanning calorimetry (DSC) and Trp fluorescence assays carried out on DPPC model membranes after incubation with huPrP60-91 showed a marked tendency of the peptide to slowly penetrate the lipid bilayer with a concomitant conformational transition toward an extended β-sheet-like structure. Such an event, which was ascribed to the hydrophobic Trp side chain residues, was shown to also depend on the level of copper(II) occupancy along the peptide. Conversely, the CD spectra of chPrP53-76 aqueous solutions indicated the presence of a mixture of random-coil/β-turn-like structures whose resulting equilibrium was influenced by SDS and copper(II) addition. Furthermore, chPrP53-76 did not exhibit any tendency to interact with model membranes in either the presence or absence of copper(II). The results reported here provide evidence of the different roles played by environmental factors in affecting the conformation and membrane activity of human and avian prion N-terminal domains.

Cross‐Talk Between the Octarepeat Domain and the Fifth Binding Site of Prion Protein Driven by the Interaction of Copper(II) with the N‐terminus

“…In a similar fashion, the structuring effect of the metal ion on the conformation of the PrP peptide fragments outside the tetraoctarepeat domain caused a decrease in the negative band, which was distinctive of the far-UV CD spectra of AcPr (106)(107)(108)(109)(110)(111)(112)(113)(114), [98] AcPrP(106-126) [98] and AcPrP [see Figures 1S a-c, respectively, in the Supporting Information). The far-UV CD spectrum of AcPrP(60-91) [103] ( Figure 1S f in the Supporting Information) was also affected by increasing amounts of copper(II), and shows an isodichroic point at l = 210 nm and an increase of the negative band centred at l = 220 nm. Nevertheless, the negative band centred below l = 200 nm shifted toward higher wavelengths and gave rise to a positive band that increased with increased metal/peptide ratio, showing a differerent trend from those observed for the other PrP peptide fragments.…”

Amino acid-based tweezers: The role of turn-like conformation in the binding of copper(II)

“…Consequently, avian prion protein unit can bind only two Cu(II) equivalents with the same geometry at any Cu(II):peptide ratio. It was speculated that conformational preferences of avian peptide contribute to the Cu(II) binding mode and maybe to the overall conformational integrity of the protein and its protection from pathogenic conversion [9,10].…”

Evolutionary Implications of Metal Binding Features in Different Species’ Prion Protein: An Inorganic Point of View

“…The different speciation, stoichiometry and binding features coming out from the potentiometric and spectroscopic studies account for the absence of the interaction of the Cu(II)-tetra-hexarepeat with membrane models, differently from that reported for the Cu(II)-tetraoctarepeat system [90]. Environmental factors, copper(II), pH and membrane-mimicking environments play a role in assisting different conformational preferences of chicken tetra-hexarepeats in comparison with the mammalian tetra-octarepeats [90].…”

“…Environmental factors, copper(II), pH and membrane-mimicking environments play a role in assisting different conformational preferences of chicken tetra-hexarepeats in comparison with the mammalian tetra-octarepeats [90]. …”