Refinement of under-determined loops of Human Prion Protein by database-derived distance constraints

Abstract

Due to insufficient experimental restraints, a biologically critical loop region in PrP(c) (residues 167-171), which is a potential binding site for Protein X, is under-determined in most mammalian species. Here, we show that by adding information about distance constraints derived from a database of high-resolution protein structures, this under-determined loop as well as other secondary structural elements of the E200K variant of Human Prion Protein (hPrP(c)), a disease-related isoform, can be refined into more realistic structures in the structural ensemble with improved quality and increased accuracy. In particular, the ensemble becomes more compact after the refinement and the percentage of residues in the most favourable region of the Ramachandran diagram is increased to about 90% in the refined structures from the 80% to 85% range in the previously reported structures.

Fig. 1

Sample distance distribution functions. (a) Distances between the C atom of ARG at position i and the O atom ILE at position i + 1. (b) Distances between the C^β atom of ALA at position i and the N atom of LEU at position i + 2.

Fig. 2

Residue-residue comparison between the refined E200K and wild-type NMR and X-ray structures. The graphs show the residue RMSD values (with the backbone atoms, N, C^α, C′, and O) for the average and energy-minimized structures of <E200K>^NMR+D (magenta line) and <E200K>^NMR (green line) compared with the structures of (a) the wild-type hPrP^c NMR structure (1QM0) at mildly acidic condition (pH 4.5); (b) the wild-type hPrP^c NMR structure (1HJM) at neutral condition (pH 7.0); (c) the wild-type shPrP^c X-ray structure (1UW3) in a monomeric form; (d) the wild-type hPrP^c X-ray structure (1I4M) in a dimeric form. The secondary structures are indicated along the top of each part of the figure with h representing alpha helix and s beta sheet.

Fig. 3

Detailed residue RMSD plots for helix and loop regions. The graphs show the detailed residue RMSD values (of the backbone atoms, N, C^α, C′, and O) for the average and energy-minimized structures of <E200K>^NMR+D (magenta line) and <E200K>^NMR (green line) at (a) N-terminal of Helix 2 (residues 172–190) when compared with the hPrP^c X-ray structure; (b) C-terminal of Helix 2 and the loop between Helix 2 and Helix 3 (residues 191–199) compared with the hPrP^c X-ray structure; and (c) Helix 3 (residues 201–228) when compared with the hPrP^c X-ray structure.

Fig. 4

The ϕ and ψ angles of the refined E200K structures. The angles of ^NMR+D are represented by magenta squares and for <E200K>^NMR by green squares.

Fig. 5

Ramachandran plots of ^NMR and <E200K>^NMR+D. (a) Ramachandran plot showing the values of (ϕ, ψ) angles of the average and energy-minimized structure of <E200K>^NMR; (b) Ramachandran plot of <E200K>^NMR+D. Only the residues of Loop 1 are shown. In (a), most of the residues in Loop 1 are found outside the most favourable (red) regions, while in (b), most of these residues lie within the most favourable regions.

Fig. 6

Superimposition of tertiary structures. Tertiary structures of the average and energy-minimized structures of ^NMR+D (small magenta cylinders) and <E200K>^NMR (small green cylinders) are superimposed in (a) the backbone of the whole protein; (b) the backbone of Loop 1; (c) the backbone of Loop 2; (d) the backbone and side-chain of Loop 1; (e) the backbone and side-chain of Loop 2.