Functional implications of multistage copper binding to the prion protein

Abstract

The prion protein (PrP) is responsible for a group of neurodegenerative diseases called the transmissible spongiform encephalopathies. The normal function of PrP has not yet been discovered, but indirect evidence suggests a linkage to its ability to bind copper. In this article, low-copper-concentration bindings of Cu 2؉ to PrP are investigated by using a recently developed hybrid density functional theory (DFT)/DFT method. It is found that at the lowest copper concentrations, the binding site consists of 4 histidine residues coordinating the copper through imidazole nitrogens. At higher concentrations, 2 histidines are involved in the binding, one of them in the axial position. These results are in good agreement with existing experimental data. Comparison of free energies for all modes of coordination shows that when enough copper is available, the binding sites will spontaneously rearrange to accommodate more copper ions, despite the fact that binding energy per copper ion decreases with concentration. These findings support the hypothesis that PrP acts as a copper buffer in vivo, protecting other proteins from the attachment of copper ions. Using large-scale classical molecular dynamics, we also probe the structure of full-length copper-bound PrP, including its unfolded N-terminal domain. The results show that copper attachment leads to rearrangement of the structure of the Cu-bonded octarepeat region and to development of turns in areas separating copperbound residues. These turns make the flexible N-terminal domain more rigid and thus more resistant to misfolding. The last result suggests that copper binding plays a beneficial role in the initial stages of prion diseases.copper attachment ͉ hybrid DFT ͉ misfolding ͉ neurodegenerative diseases ͉ metalloprotein

Copper Alters Aggregation Behavior of Prion Protein and Induces Novel Interactions between Its N- and C-terminal Regions

“…We did not observe any change in thioflavin T fluorescence with Cu 2ϩ -bound PrP(23-231) at various temperatures (16,20,25,30, and 37°C), indicating that the aggregates formed are amorphous in nature (supplemental Fig. S2B).…”

“…Cu 2ϩ -binding induces conformational changes leading to reduction in the helical content and an increase in the ␤-sheet content especially in the region 90 -120 (13), which partially overlaps with the well established amyloidogenic stretch 106 -126 (14). Cu 2ϩ has also been shown to induce conformational changes and increased stability in the octapeptide repeats of PrP (15,16). Cu 2ϩ is shown to convert PrP C from brain homogenates (2) and aged recombinant PrP (17) to protease-resistant and detergent-insoluble aggregates.…”

“…Conformational studies on binding of Cu 2ϩ to PrP in vitro have been performed using peptides (26 -28) or truncated variants of PrP (29 -31) and at nonphysiological temperatures (2,10,(13)(14)(15)(16)(17) and in water or buffers of low ionic strength (3,9,13,(32)(33)(34). We have used full-length PrP to investigate conformational changes and aggregation upon Cu 2ϩ -binding at physiological temperature (37°C).…”

Applications of large-scale density functional theory in biology

“…The resulting approach has been demonstrated in ab initio MD simulations [50]. RMGDFT, in particular, has been applied to various biological systems, including prion folding [51,52], but this has usually been as part of a mixed scheme involving embedding a relatively small full-DFT region within an orbital-free DFT treatment of a surrounding environment. There are also grid-based codes with strengths in particular domains such as modelling excited states and spectroscopy, including OCTOPUS [53].…”

Redox Control of Prion and Disease Pathogenesis

“…Some of the contradictory results pertaining to beneficial or harmful effects of copper noted earlier can be explained by observations from in vitro studies in which copper inhibits amplification of PrP Sc from purified brain-derived and recombinant PrP C by stabilizing the structure of the latter (93,143). At the same time, copper is known to enhance the b-sheet structure when added to preformed PrP fibrils, increasing their overall PrP Sc content (21).…”