Local Structural Plasticity of the Prion Protein. Analysis of NMR Relaxation Dynamics

Abstract

A template-assisted conformational change of the cellular prion protein (PrP(C)) from a predominantly helical structure to an amyloid-type structure with a higher proportion of beta-sheet is thought to be the causative factor in prion diseases. Since flexibility of the polypeptide is likely to contribute to the ability of PrP(C) to undergo the conformational change that leads to the infective state, we have undertaken a comprehensive examination of the dynamics of two recombinant Syrian hamster PrP fragments, PrP(29-231) and PrP(90-231), using (15)N NMR relaxation measurements. The molecular motions of these PrP fragments have been studied in solution using (15)N longitudinal (T(1)) and transverse relaxation (T(2)) measurements as well as [(1)H]-(15)N nuclear Overhauser effects (NOE). These data have been analyzed using both reduced spectral density mapping and the Lipari-Szabo model free formalism. The relaxation properties of the common regions of PrP(29-231) and PrP(90-231) are very similar; both have a relatively inflexible globular domain (residues 128-227) with a highly flexible and largely unstructured N-terminal domain. Residues 29-89 of PrP(29-231), which include the copper-binding octarepeat sequences, are also highly flexible. Analysis of the spectral densities at each residue indicates that even within the structured core of PrP(C), a markedly diverse range of motions is observed, consistent with the inherent plasticity of the protein. The central portions of helices B and C form a relatively rigid core, which is stabilized by the presence of an interhelix disulfide bond. Of the remainder of the globular domain, the parts that are not in direct contact with the rigid region, including helix A, are more flexible. Most significantly, slow conformational fluctuations on a millisecond to microsecond time scale are observed for the small beta-sheet. These results are consistent with the hypothesis that the infectious, scrapie form of the protein PrP(Sc) could contain a helical core consisting of helices B and C, similar in structure to the cellular form PrP(C). Our results indicate that residues 90-140, which are required for prion infectivity, are relatively flexible in PrP(C), consistent with a lowered thermodynamic barrier to a template-assisted conformational change to the infectious beta-sheet-rich scrapie isoform.

Molecular Dynamics as an Approach to Study Prion Protein Misfolding and the Effect of Pathogenic Mutations

“…The flexibility of the S2-HB and HB-HC loops is high, in accordance with experimental data [84,85]. Although the capping box at the N-terminal end of HC was not present in the starting structure (obtained from the representative NMR structure with PDB code 1QLX), it formed within 1 ns in all simulations at neutral pH.…”

“…NMR relaxation studies on hamster and mouse PrP further indicate fast picosecond time-scale motions of the backbone amides from res. 222 [84,85].…”

“…A large effect on the conformation and dynamics can therefore be expected. Notably, changes to the most stable part of the PrP structure (as determined by NMR and hydrogen exchange [39,76,79,85]) are minimal (Fig. 2).…”

Dynamics of a truncated prion protein, PrP(113–231), from 15N NMR relaxation: Order parameters calculated and slow conformational fluctuations localized to a distinct region

“…All mammalian PrP C studied consist of two structurally distinct domains. The N-terminal domain (residues 23-120) is highly disordered 15 and is notable for its ability to bind Cu 2þ ions. [16][17][18][19][20][21] The N-terminus contains a highly conserved octa-repeating sequence, PHGGGWGQ between residues 57 and 90.…”

“…15 N relaxation measurements have previously been reported for full-length PrP 15 and the 90-231 fragment. 15,31,32 However, attempts to obtain order parameters (S 2 ) for each NHbond vector have been unsuccessful for the majority of residues in helix B and C for these PrP C constructs. 15 These fragments contain a significant amount of the unstructured tail.…”