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Comparative Study
. 2009 Apr;109(1):60-73.
doi: 10.1111/j.1471-4159.2009.05892.x. Epub 2009 Feb 23.

Characterization of cell-surface prion protein relative to its recombinant analogue: insights from molecular dynamics simulations of diglycosylated, membrane-bound human prion protein

Mari L DeMarco  1 , Valerie Daggett
Affiliations
Comparative Study

Characterization of cell-surface prion protein relative to its recombinant analogue: insights from molecular dynamics simulations of diglycosylated, membrane-bound human prion protein

Mari L DeMarco et al. J Neurochem. 2009 Apr.

Abstract

The prion protein (PrP) is responsible for several fatal neurodegenerative diseases via conversion from its normal to disease-related isoform. The recombinant form of the protein is typically studied to investigate the conversion process. This constructs lacks the co- and post-translational modifications present in vivo, there the protein has two N-linked glycans and is bound to the outer leaflet of the plasma membrane via a glycosylphosphatidylinositol (GPI) anchor. The inherent flexibility and heterogeneity of the glycans, the plasticity of the GPI anchor, and the localization of the protein in a membrane make experimental structural characterization of biological constructs of cellular prion protein (PrP(C)) challenging. Yet this characterization is central in determining not only the suitability of recombinant (rec)-PrP(C) as a model for biological forms of the protein but also the potential role of co- and post-translational modifications on the disease process. Here, we present molecular dynamics simulations of three human prion protein constructs: (i) a protein-only construct modeling the recombinant form, (ii) a diglycosylated and soluble construct, and (iii) a diglycosylated and GPI-anchored construct bound to a lipid bilayer. We found that glycosylation and membrane anchoring do not significantly alter the structure or dynamics of PrP(C), but they do appreciably modify the accessibility of the polypeptide surface PrP(C). In addition, the simulations of membrane-bound PrP(C) revealed likely recognition domains for the disease-initiating PrP(C):PrP(Sc) (infectious and/or misfolded form of the prion protein) binding event and a potential mechanism for the observed inefficiency of conversion associated with differentially glycosylated PrP species.

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Figures

Figure 1
Figure 1
The starting structures for the PrPC simulations: PrPrec, PrPglyco and PrPgpi. The structures are colored as follows: unstructured N-terminus, residues 90-109 (green), β-strands S1 and S2 (magenta), helices HA (light blue), HB and HC (blue), glycan-1 (orange), glycan-2 (purple), GPI anchor (black), POPC bilayer (gray). Note that actual simulations contain explicit water molecules but they are not displayed here.
Figure 2
Figure 2
Sequence and linkage information for the models of the (A) 13-residue PrPC glycan attached at Asn-181 and Asn-197, and (B) the GPI anchor linked to Ser-230.
Figure 3
Figure 3
The dynamics of the different PrP constructs. (A) Cα RMSD of the globular region of PrP (residues 125-228). (B & C) Average Cα RMSF of each residue over the course of each 15 ns simulation.
Figure 4
Figure 4
Snapshots from the PrPglyco neutral (A) and low pH (B) and the PrPgpi neutral (C) and low pH (D) simulations. Colored as in Figure 1.
Figure 5
Figure 5
The number of glycan-protein and GPI-protein atom-atom contacts as a function of time for PrPglyco (A & B) and PrPgpi (C & D) simulations: glycan-1 (orange), glycan-2 (purple) and GPI-achor (black).
Figure 6
Figure 6
Volumes occupied by glycan-1 (orange) and glycan-2 (purple) from the PrPglyco simulations. Overlay of glycans taken at 1 ns intervals from 0-15 ns, mapped onto the 15 ns structures.
Figure 7
Figure 7
Volumes occupied by glycan-1 (orange) and glycan-2 (purple) over the course of the PrPgpi simulations. (A & B) Overlay of glycans taken at 1 ns intervals from 0-15 ns, mapped onto the 15 ns structure, and (C & D) the same image as viewed from the top of the bilayer.
Figure 8
Figure 8
The percentage of time (over the course of the simulations) that glycan and/or GPI anchor atoms were in contact with the polypeptide of PrPglyco (A & B), and PrPgpi (C & D); mapped onto the corresponding final structures from simulation.
Figure 9
Figure 9
Surface properties of the polypeptide from cell-surface PrPC relative to its recombinant analogue. To approximate accessibility of the surface to receptor molecules, a probe with a 4.0 Å radius was used to calculate surface accessibility. (A-D) Accessible PrP surfaces not affected by the presence of the glycans, GPI anchor or lipid bilayer (cyan) for the neutral (A & B) and low pH (C&D) simulations of PrPgpi. (E) Experimentally determined antibody epitopes accessible in both recombinant and cell-surface PrPC (blue). (F) Experimentally determined epitope accessible in recombinant but not cell-surface PrPC (red). (G) Residues (109-141, 166-179, 200-223) that correspond to peptides that bind PrPC and inhibit conversion (green). (H) Residues critical to species barrier (orange). (I) Human disease-related mutations and the M129V polymorphism (pink).
Figure 10
Figure 10
Conversion inefficiencies associated with glycosylation may be due in part to interactions between HA (light blue) and glycan-2 (purple). As previously demonstrated, the conversion of huPrPCrec → PrPScrec is facilitated by the mobility of HA (DeMarco and Daggett 2007).
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