Identification of a Conserved N-Capping Box Important for the Structural Autonomy of the Prion α3-Helix: The Disease Associated D202N Mutation Destabilizes the Helical Conformation

Abstract

Peptides corresponding to three alpha helices present in the C-terminal region of the human prion protein have been synthesized and their structural autonomy analyzed by circular dichroism (CD) and NMR spectroscopy. The results obtained indicate that the protein fragment corresponding to the α3-helix, in contrast to α1 and α2 peptides, shows a complete structural autonomy. The chemical shifts values found for NH and CHa resonances of the isolated α3 peptide, formed by 30 aminoacid residues, were markedly and surprisingly similar to the corresponding values of the α3-helix in the protein. The structural autonomy of the α3-helix is profoundly determined by the presence of the conserved capping box and, in part, by the ionic bond formed between Glu200 and Lys204. On the basis of these observations a novel PrP consensus pattern, centered on the α3-helix region, has been defined. The data indicate that this autonomous and highly conserved region of the PrP^c likely plays a critical role in folding and stability. This gives an explanation of why many of pathogenic mutations occur in this part of the molecule, sharing relevant effects on the overall protein conformation. In particular the D202N capping mutation almost completely destabilizes the isolated α3 peptide. While it is well known that the D202N substitution is associated with a GSS disease, the possible structural basis of this fatal pathology has never been investigated. We propose that a lower α3-helical propensity leading to a major destabilization of the PrP^c molecule initiates the pathogenic process associated with D202N capping mutation.