On the structural stability of the free and metal-loaded c-terminal domain of the prion protein

A misfolded conformer of the cellular prion protein, denoted as scrapie prion protein, is considered responsible for a variety of fatal neurodegenerative diseases. Both, the function of the protein in its native conformation as well as the factors that trigger the protein misfolding and its mechanism remain as yet open issues. The aim of this thesis is to give a contribution to answering these questions by means of a variety of tools currently available in the computational (bio)chemistry community. The capability of the cellular prion protein isoform to bind metal is supported by controversial evidences and atomistic details of putative binding sites are missing. Based on the NMR structure of the C-terminal part of the cellular isoform, we performed a hybrid quantum/classical (QM/MM) Car-Parrinello molecular dynamics study to identify likely Cu(II) binding sites. By means of a bioinformatics approach, we localized the protein regions with the highest propensity for copper ion binding. The identified candidate structures were subsequently refined via QM/MM simulations and then probed by computing their EPR characteristics. Overall best agreement with the experimental EPR data was observed for a binding site involving H187. The previously characterized prion protein Cu(II)-binding sites were substituted for Mn(II) and Zn(II) and subsequently refined by means of the QM/MM Car-Parrinello approach. As a general conclusion, all the Cu-binding sites lead to stable arrangements upon Mn and Zn substitution with relative minor structural changes even within the ps time scale. The validity of the binding motifs obtained was assessed by means of comparison with a pool of known Mn and Zn binding proteins and common features were found and described. The Zn binding site in proximity of H177 was speculated to act as a protein interface zinc site with a possible direct involvement in the aggregation process. The EPR parameters of the Mn-containing binding sites were also computed. The influence of some environmental factors that can possibly trigger the misfolding event were investigated via classical molecular dynamics simulations. Specifically, protein solution containing a monovalent salt (NaCl) in four different concentrations (0.0, 0.05, 0.1, 0.2 M) were simulated at three different pH (pH 2, 4, 7) in order to highlight structural changes and local weaknesses in the protein native structural motifs. The effects of the ionic strength and pH on the secondary structural elements of the protein were identified and characterized whereas the main topological framework was preserved in all simulations. The energy landscape of the cellular to scrapie isoform misfolding pathway is currently under investigation by means of a parallel tempering (replica exchange) enhanced sampling technique in combination with classical molecular dynamics. The screening of the data collected led to the identification of a pool of prion protein scrapie candidates. A set of new folding motifs were characterized. Preliminary results and plans for future investigations are presented. With the goal of providing a structural model for the minimal misfolded aggregate based on the most likely prion protein scrapie candidates, a protein-protein docking approach was used to generate protofibril models and their structural stability and transformations investigated by means of classical molecular dynamics simulations.

Röthlisberger, Ursula
Lausanne, EPFL
Other identifiers:
urn: urn:nbn:ch:bel-epfl-thesis3592-7

Note: The status of this file is: EPFL only

 Record created 2006-06-13, last modified 2018-03-17

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