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. 2000 Jul 18;97(15):8340-5.
doi: 10.1073/pnas.97.15.8340.

NMR structures of three single-residue variants of the human prion protein

L Calzolai  1 , D A Lysek, P Guntert, C von Schroetter, R Riek, R Zahn, K Wüthrich
Affiliations

NMR structures of three single-residue variants of the human prion protein

L Calzolai et al. Proc Natl Acad Sci U S A. .

Abstract

The NMR structures of three single-amino acid variants of the C-terminal domain of the human prion protein, hPrP(121-230), are presented. In hPrP(M166V) and hPrP(R220K) the substitution is with the corresponding residue in murine PrP, and in hPrP(S170N) it is with the corresponding Syrian hamster residue. All three substitutions are in the surface region of the structure of the cellular form of PrP (PrP(C)) that is formed by the C-terminal part of helix 3, with residues 218-230, and a loop of residues 166-172. This molecular region shows high species variability and has been implicated in specific interactions with a so far not further characterized "protein X," and it is related to the species barrier for transmission of prion diseases. As expected, the three variant hPrP(121-230) structures have the same global architecture as the previously determined wild-type bovine, human, murine, and Syrian hamster prion proteins, but with the present study two localized "conformational markers" could be related with single amino acid exchanges. These are the length and quality of definition of helix 3, and the NMR-observability of the residues in the loop 166-172. Poor definition of the C-terminal part of helix 3 is characteristic for murine PrP and has now been observed also for hPrP(R220K), and NMR observation of the complete loop 166-172 has so far been unique for Syrian hamster PrP and is now also documented for hPrP(S170N).

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Figures

Figure 1
Figure 1
Amino acid sequence alignment of the fragment 125–230 (numeration of hPrP following ref. ; for each protein the number in parentheses on the right indicates the sequence position attributed to the C-terminal residue) of the human, bovine, murine, and Syrian hamster PrPs. At the top the locations of the regular secondary structure elements of hPrP(121–230) are indicated. The red letters identify residue positions with amino acid exchanges or insertions in the region of the three-dimensional structure that are discussed in this paper. The row “hPrP var” lists the amino acid exchanges in the single-residue variants of hPrP studied in this paper, where the doubly underlined residues indicate those variants for which a complete structure determination is presented. In the row “factor X” the black circles identify the sequence positions 168, 172, 215, and 219, which have been suggested to form an epitope or part of an epitope for interactions with a species-specific “factor X” or “protein X” (15, 33).
Figure 2
Figure 2
Two-dimensional [15N,1H]-COSY spectra. (a) hPrP(S170N). (b) hPrP(121–230). A spectral region is shown that contains most of the resonances of the loop 166–172 in hPrP(S170N). The following notation is used for the peak assignments: Cross peaks detected in both proteins are black. Cross peaks present only in hPrP(S170N) are red. Cross peaks belonging to residues 166–175 are bold. The rectangular frames display cross peaks outside of the region shown here, with the chemical shifts of the peaks indicated in italics. The spectra were recorded at 600 MHz with 1 mM protein solutions at pH 4.5 and 20°C.
Figure 3
Figure 3
Superposition of the polypeptide backbone from residues 125–230 in different PrP NMR structures for best fit of the backbone heavy atoms of residues 125–220. The radius of the cylindrical rods representing the polypeptide chains is proportional to the mean global backbone displacement per residue (35) among the 20 energy-minimized conformers used to represent the NMR structures. The views on the right were obtained from those on the left by a −90° rotation about a vertical axis. (a) hPrP(121–230) (red) and mPrP(121–231) (yellow). (b) hPrP(121–230) (red) and hPrP(R220K) (cyan). (c) hPrP(121–230) (red) and hPrP(M166V) (turquoise). (d) hPrP(121–230) (red) and hPrP(S170N) (amber). For these comparisons we recalculated the NMR structures of mPrP(121–231) (2) and hPrP(121–230) (6), using the same protocol as for the hPrP(121–230) variants (see Materials and Methods).
Figure 4
Figure 4
Comparison of helix 3 in hPrP(121–230) (red), mPrP(121–231) (yellow), and hPrP(R220K) (cyan) with the NMR structures represented by bundles of 20 conformers (28). (a) Polypeptide backbone of residues 200–229 superimposed for best fit of the residues 200–218. (b) Residues 213–229 superimposed for best fit of the residues 221–225.
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