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From: TSS (
Subject: Re: Prion protein NMR structures of chickens, turtles, and frogs
Date: January 18, 2005 at 1:43 pm PST

In Reply to: Prion protein NMR structures of chickens, turtles, and frogs posted by TSS on January 17, 2005 at 1:05 pm:

Prion protein NMR structures of chickens, turtles,
and frogs

Luigi Calzolai*†‡, Dominikus A. Lysek*†, Daniel R. Pe´ rez*†§, Peter Gu¨ ntert*¶, and Kurt Wu¨ thrich*
*Institut fu¨ r Molekularbiologie und Biophysik, Eidgeno¨ ssische Technische Hochschule Zu¨ rich, CH-8093 Zu¨ rich, Switzerland; and §Graduate Program in
Molecular, Cellular, and Neuroscience Biology, Facultad de Ciencias, Universidad de Chile, Casilla 653, Santiago, Chile
Contributed by Kurt Wu¨ thrich, December 6, 2004
The NMR structures of the recombinant prion proteins from
chicken (Gallus gallus; chPrP), the red-eared slider turtle (Trachemys
scripta; tPrP), and the African clawed frog (Xenopus laevis;
xlPrP) are presented. The amino acid sequences of these prion
proteins show 30% identity with mammalian prion proteins. All
three species form the same molecular architecture as mammalian
PrPC, with a long, flexibly disordered tail attached to the N-terminal
end of a globular domain. The globular domain in chPrP and tPrP
contains three -helices, one short 310-helix, and a short antiparallel
-sheet. In xlPrP, the globular domain includes three -helices
and a somewhat longer -sheet than in the other species. The
spatial arrangement of these regular secondary structures coincides
closely with that of the globular domain in mammalian prion
proteins. Based on the low sequence identity to mammalian PrPs,
comparison of chPrP, tPrP, and xlPrP with mammalian PrPC structures
is used to identify a set of essential amino acid positions for
the preservation of the same PrPC fold in birds, reptiles, amphibians,
and mammals. There are additional conserved residues without
apparent structural roles, which are of interest for the ongoing
search for physiological functions of PrPC in healthy organisms.
nonmammalian species  transmissible spongiform encephalopathy
The prion protein (PrP) has attracted a lot of interest because
of its relation to transmissible spongiform encephalopathies
(TSEs), which are a group of invariably fatal neurological
diseases (1). Healthy organisms that do not express a prion
protein, such as suitably selected transgenic laboratory animals,
cannot develop a TSE (2), and the ‘‘protein-only hypothesis’’
further attributes TSE-causing infectivity to an aggregated
‘‘scrapie form’’ of PrP (PrPSC) that has been isolated from brain
tissue of diseased organisms (1). Although TSEs have only been
documented for mammalian species, PrP has been identified in
a wider range of higher organisms, which on an evolutionary
scale extends at least down to amphibians (3–9). In apparent
contrast to the high sequence conservation among mammalian
PrPs, no physiological function has been reliably attributed to the
‘‘cellular form’’ of PrP (PrPC) found in healthy organisms.
In view of its critical role in TSEs, the prion protein has also
attracted considerable interest by structural biologists. So far,
atomic resolution structure determination was focused on recombinant
mammalian prion proteins (10–18), which have recently
been shown to represent the protein architecture of PrPC
(19). As a group, the mammalian PrPs have 90% sequence
identity (4). Here, we present the NMR structures of recombinant
PrP from chicken, turtle, and frog (chPrP, tPrP, and xlPrP,
respectively), each of which has 30% sequence identity with
mammalian PrP. We then exploit this low homology in searches,
based on comparison of the three-dimensional structures, for
conserved amino acids with apparent roles in maintaining a
common PrPC-fold, and for nonstructural conserved amino acids
that might provide leads to the unknown physiological function
of PrPC.
Materials and Methods
Protein Preparation. The chPrP gene was provided to us by D. A.
Harris (Washington University School of Medicine, St. Louis),
the tPrP gene was given to us by T. Simonic (Universita` di
Milano, Milano, Italy), and the xlPrP gene was obtained from a
cDNA library. All three genes were cloned into the vector
pRSETA, and the proteins were expressed in Escherichia coli.
For the purification of the recombinant proteins, we followed
described procedures (20).
NMR Measurements and Structure Calculations. NMR measurements
were performed at 20°C on Bruker DRX500, DRX600,
and DRX750 and Avance900 spectrometers. The protein samples
used were uniformly 15N-labeled and 13C,15N-labeled
tPrP(23–225), tPrP(121–225), chPrP(23–225), chPrP(121–225),
and xlPrP(90–222). In this notation, the numbers in parentheses
identify the first and last residues in the individual constructs,
and the numeration for human PrP is used as explained in the
caption to Fig. 1. The NMR samples were in ether 95% H2O5%
D2O or 99.9% D2O, contained 5 mM sodium acetate buffer at
pH 4.5 (pH 4.3 for chPrP), and the protein concentration was
0.6–1.0 mM. The programs PROSA (21) and XEASY (22) were
used for data processing and spectral analysis, respectively.
Sequence-specific resonance assignments for the proteins were
obtained by using standard triple-resonance NMR experiments
(23). Distance constraints for the input for the structure
determination were obtained from three-dimensional 13Cresolved
[1H,1H]-NOESY, three-dimensional 15N-resolved
[1H,1H]-NOESY, and two-dimensional [1H,1H]-NOESY spectra
recorded at a proton frequency of 750 or 900 MHz with mixing
times of 40 or 50 ms. The automatic nuclear Overhauser
enhancement (NOE) assignment module CANDID (24) implemented
in the program DYANA (25) was used for the structure
calculations. DYANA was also used to convert NOE intensities
into upper distance constraints according to an inverse sixth
power peak volume-to-distance relationship, to remove meaningless
constraints, and to derive constraints for the backbone
torsion angles and from the C chemical shift values (26, 27).
The final round of structure calculation with DYANA was started
with 100 randomized conformers. The 20 conformers with the
lowest final target function values were energy-minimized in a
water shell with in the program OPALP (28, 29) using the AMBER
force field (30). The program MOLMOL (31) was used to analyze
the results of the structure calculations (Table 1) and to prepare
the drawings of the structures.
Abbreviations: PrP, prion protein; TSE, transmissible spongiform encephalopathy; PrPC,
cellular PrP; chPrP, chicken PrP; tPrP, turtle PrP; xlPrP, frog PrP.
Data deposition: The sequences reported in this paper have been deposited in the Protein
Data Bank, [PDB ID codes 1U3M for chPrP(121–225), 1U5L for tPrP(121–225),
and 1XU0 for xlPrP(90 –222)].
†L.C., D.A.L., and D.R.P. contributed equally to this work.
‡Present address: Department of Biotechnology and Molecular Sciences, University of
Insubria, Via J. Dunant 3, 21100 Varese, Italy.
¶Present address: RIKEN Genomic Sciences Center, 1-7-22 Suehiro, Tsurumi, Yokohama
231-0045, Japan.
To whom correspondence should be addressed. E-mail:
© 2005 by The National Academy of Sciences of the USA
www.pnas.orgcgidoi10.1073pnas.0408939102 PNAS  January 18, 2005  vol. 102  no. 3  651–655
Database Searches. The SwissProt and TrEMBL databases (32)
of proteins were searched by using the PATTINPROT tool at the
Pole Bio-Informatique Lyonnaise ( The
databases were searched by using the query E-x (2)-[WY]-
The NMR structures of recombinant chPrP(23–225), tPrP(23–
225), and xlPrP(99–222) (see Fig. 1 for the sequence numeration
used) all contain a globular domain comprising a Cterminal
segment of 100 residues. N-terminal to this
structured domain there is a f lexibly disordered tail of 110
residues for chPrP(23–225), 118 residues for tPrP(23–225), and
69 residues for mature xlPrP (Fig. 1). These global architectures
coincide with those of mammalian PrPs(10–18), and they
were clearly evidenced by different dispersion of the 1H
chemical shifts and different values of the steady-state
15N{1H}-NOEs for the tail and the globular domain in each
Fig. 1. Sequence alignment of hPrP, chPrP, tPrP, and xlPrP, where hPrP represents ‘‘mammalian-type PrP.’’ At the top is the residue numbering for hPrP, which
is used throughout this manuscript, i.e., insertions and deletions in chPrP, tPrP, and xlPrP required for maximal coincidence with hPrP are not consecutively
numbered. In this alignment, the amino acids shown in red are identical in all four PrPs, and the ones displayed in blue are identical in chPrP, tPrP, and hPrP. The
black box identifies the segment containing polypeptide repeats (see text) for which no individual alignments were attempted. The GPI attachment site is
identified with a green box, and the glycosylation sites (asparagine attachment site and nearest-next threonineserine) is identified with light blue boxes. The
orange boxes indicate segments with higher than average sequence conservation that do not have an apparent stabilizing role in the PrP fold, and might thus
be conserved for functional reasons. These include the polypeptide segment 23–42 with the N-terminal signaling-peptide cleavage site, which has been
suggested to be the signaling-peptide for reinternalization of PrP (36), a Src homology 3 (SH3)-binding motif of residues 100–110 (37, 38), the segment 113–128
representing a predicted transmembrane helix (39, 40), and the segment 146–155 in hPrP, chPrP, and tPrP that shows similarity to the laminin 2-chain (see text).
The pink boxes indicate segments with high amino acid identity that appear to be needed for the stability of the ‘‘PrP-fold’’ (see text). At the bottom, the regular
secondary structures in the globular domain of the four proteins are indicated. The sequence alignment was performed interactively so as to align a maximal
number of identical residues. For the globular domain (121–230), the alignment was also based on visual inspection of the three-dimensional structures. In chPrP,
the insertion at the end of helix 2 was divided into two segments to properly align the glycosylation site 197–199.
Table 1. Input for the structure calculation and characterization of the energy-minimized NMR solution structures
of tPrP(121–225), chPrP(121–225), and xIPrP(90–222)
Quantity* chPrP(121–225) tPrP(121–225) xIPrP(90–222)
NOE upper distance limits 1,889 1,522 2,283
Dihedral angle constraints 123 110 –
Residual target function value, Å2 1.62  0.20 1.75  0.18 1.99  0,19
Residual distance constraint violations
Number  0.1 Å 29 2 304 24 4
Maximum, Å 0.16  0.03 0.14  0.01 0.14  0.01
Residual dihedral angle constraint violations
Number  2.0° 1 1 0 –
Maximum, ° 2.13  0.80 1.21  0.71 –
Amber energies, kcalmol
Total 4,633  98 4,694  69 5,215  60
van der Waals 285  17 309  16 244  17
Electrostatic 5,285  94 5,265  74 6,032  59
rms deviation to the averaged coordinates (Å)†
Backbone (N, C, C) 0.72  0.14
(135–200, 211–237)
0.85  0.13
0.75  0.13
(127–166, 172–225)
All heavy atoms 1.17  0.13 1.33  0.15 1.19  0.12
*Except for the toptwoentries, the average for the 20 conformers with the lowest residual DYANA target function values and the standard
deviation among them are given. NOE, nuclear Overhauser enhancement.
†The numbers in parentheses identify the polypeptide segments for which the rms deviation was calculated.
652  www.pnas.orgcgidoi10.1073pnas.0408939102 Calzolai et al.
protein. Here, we focus on the NMR structure determination
of the globular domain in the three prion proteins.
TheNMRstructures of the globular domains were determined
in the constructs chPrP(121–225), tPrP(121–225), and xlPrP(90–
222). Nearly complete resonance assignments were obtained,
and chemical shift lists have been deposited in the BioMagRes-
Bank (33–35). The structure determination protocols are presented
in Table 1. The three structures are shown in Fig. 2 B–D.
In the orientation of the proteins in Fig. 2, the N terminus is on
the right, pointing to the lower right corner, and the C terminus is
in the upper right corner. The regular secondary structures (Fig. 1)
are arranged in the three-dimensional structure as follows: The
strand 1 runs from the lower right to the upper left immediately
after the N terminus, helix 1 is in the upper left corner and
propagates toward the center of the structure, where the polypeptide
chain then forms the 2-strand of the antiparallel -sheet. A
loop of residues 166–175 on the extreme right leads to the start of
the helix 2, which ends at the lowest point of the structure. Loops
of variable length in the different proteins (Fig. 1) lead from the end
of 2 to the start of helix 3 on the left of the structure, from where
3 propagates to theCterminus in the upper right corner. In chPrP,
insertion of 11 residues in the loop linking the helices 2 and 3
(Fig. 1) is accommodated in a disordered spatial arrangement of
this polypeptide segment (Fig. 2B). Each structure has a well
ordered core of hydrophobic side chains, which are conserved
among the four species (Figs. 1 and 2).
In this section, we conduct searches for possible correlations
between amino acid sequence (Fig. 1), three-dimensional structure
of the PrPC form of the proteins in Fig. 2, and functional
properties of prion proteins. For the unstructured N-terminal
tail, these considerations are based primarily on the primary
structures (Fig. 1) and on literature data on the biochemistry and
cell biology of PrPC. For the globular domain, most of the
conclusions result from detailed comparison of the available
mammalian and nonmammalian PrPC structures (Fig. 2).
Sequence Conservation in the N-terminal Flexible Tail and PrPC Trafficking.
The prion protein sequences from mammals (represented
here by human PrP, hPrP), chicken, turtle, and frog
show several regions of above-average identity (Fig. 1). In the
N-terminal f lexibly disordered part of the protein, this includes
a glycine-rich and positively charged stretch of 15–20
residues at the chain end, which triggers the cleavage of the
N-terminal signal sequence 1–22, and has been implicated in
subcellular trafficking of PrPC (36). A highly conserved region
of residues 100–110 forms a Src homology 3 (SH3)-binding
motif, which in mammals binds the C-terminal SH3 domain of
Grb2 (37, 38). Another highly conserved segment of residues
113–128 represents a putative transmembrane helix (39, 40);
part of this segment is deleted in xlPrP (Fig. 1). There is also
the region made up of polypeptide repeats, with octarepeats in
mammalian PrP, hexarepeats in bird and reptile PrP, and no
readily apparent repeat pattern in xlPrP (Fig. 1). The tentative
functional assignments for these highly conserved sequence
elements are further validated by the three-dimensional structures
to the extent that in all of the different species they are
located in the f lexibly disordered, highly solvent-accessible
Fig. 2. NMR structures of the globular domains of hPrP, chPrP, tPrP, and
xlPrP. The polypeptide backbone fold for the residues 126–230 (see Fig. 1 for
the sequence numbering used) and the core side chains with 20% solvent
accessibility are shown for each species, as a superposition of the 20 conformers
used to represent the NMR structure. The following side chains are included
(see Fig. 1 for the sequence information): 141, 149 (only for chPrP and
xlPrP), 150 (only for hPrP and xlPrP), 161, 162, 164 (only for xlPrP), 175, 176, 179,
180 (only for hPrP, chPrP, and tPrP), 183, 184, 205, 206, 209, 210, 213, 214, and
218. (A) hPrP (side chains shown in pink). (B) chPrP (side chains shown in blue).
(C) tPrP (side chains shown in green). (D) xlPrP (side chains shown in yellow).
Calzolai et al. PNAS  January 18, 2005  vol. 102  no. 3  653
‘‘tail.’’ The most clear-cut implication results with regard to
trafficking of PrPC, because the structural elements known to
be important for cellular trafficking are highly conserved in
the PrP sequences (Fig. 1) and the PrPC three-dimensional
structures. These are the N-terminal signaling sequence (not
shown in Fig. 1) that directs PrP to the endoplasmatic reticulum,
the C-terminal signaling sequence that attaches to the
glycosylphosphatidylinositol (GPI) anchor and directs PrPC to
lipid rafts within the membrane (41), and sequence 23–42,
which directs the prion protein to clathrin-coated pits or
caveolae like domains, and therefore controls the endocytosis
of the prion protein (42).
Comparison of the Globular Domains of chPrP, tPrP, and xlPrP with
hPrP. The three-dimensional structures of the globular domains
determined in hPrP(121–230) (13), chPrP(121–225), tPrP(121–
225), and xlPrP(90–222) clearly show extensive similarities.
These similarities include the sequence locations of regular
secondary structures (Fig. 1), the three-dimensional arrangement
of the secondary structure elements (Fig. 2), and the
presence of a hydrophobic core of amino acid side chains with
low solvent accessibility (Fig. 2). In Fig. 3, this visual impression
of structural similarity is substantiated with a more quantitative
and detailed comparison.
A tight superposition of the regular secondary structures in
the four proteins shown in Fig. 2 is obtained if the helix 1 is not
included in the fitting process. The backbone heavy atoms of the
helices 2 and 3 and the -sheet are then superimposable for
any pair of the four PrPs with rms deviation values of 1.1 Å or
less (Fig. 3). The 30% identity and 50% conservation of the
sequence (Fig. 1) is then sufficient to ensure identical hydrophobic
core packing (Fig. 2). When using this approach for
structure superposition, the surface loops linking the regular
secondary structures and the orientation of helix 1 show
significant variation in the different PrPs (Fig. 3). This variation
coincides with low amino acid conservation in the loops of the
globular domain (Fig. 1). Some of these variations could be
regarded as ‘‘structural signatures’’ for PrPs from the different
evolutionary subgroups. A first example is loop 166–173, which
shows dynamic disorder in hPrP (13), whereas it is more precisely
defined in the other three proteins of Fig. 2. This loop appears
to be stabilized by a long-range hydrogen bond between V171
and Y222 in tPrP, insertion of a proline in chPrP, and a 2-aa
insertion in xlPrP. A second example is the C-terminal end of
Fig. 4. Structural features proposed to be essential for a stable PrPC fold.
Helices are shown as cylinders, with 1 shown in green and 2 and 3 shown
in dark blue. The -sheet is represented by green arrows. The single disulfide
bond is shown in yellow, and a hydrogen bond between O of T183 and the
backbone amide proton of Y162 is shown as a dashed cyan line. The backbone
segments linking these structural features are shown as a gray spline function
through the C positions. If the other elements shown are superimposed for
best fit, the helix 1 adopts somewhat different orientations in the four
proteins of Fig. 1.
Fig. 5. Conserved structural motif on the surface of the globular domains of
chPrP, tPrP, and hPrP. There is no apparent role in stabilization of the protein
fold, indicating that this motif might be conserved for reasons of the protein
function. hPrP, chPrP, and tPrP have been superimposed for local best fit of the
backbone atoms of residues 146–153, which form the helix 1 (the backbone
is shown as a gray spline function). (A) With the side chains of the residues
E146, E152, and N153. (B) With the side chains of the residues 149 and 150 (W
or Y in the different species; see Fig. 1). hPrP side chains are shown in pink,
chPrP side chains are shown in blue, and tPrP side chains are shown in green.
Fig. 3. Comparison of the three nonmammalian PrPs investigated here with
hPrP, representing the ‘‘mammalian-type’’ PrPC fold. Pairwise superpositions
of the polypeptide backbone folds are shown. (A) chPrP(121–225) (blue) and
hPrP(121–230) (red). (B) tPrP(121–225) (green) and hPrP(121–230) (red). (C)
xlPrP(90–222) (cyan) and hPrP(121–230) (red). The protein fragments of residues
128–222 are displayed. The proteins were aligned for best fit of segments
128–131, 161–164, 173–187, and 201–222. The views on the right relate to
those on the left by a 180° rotation about a vertical axis.
654  www.pnas.orgcgidoi10.1073pnas.0408939102 Calzolai et al.
helix 2, which consists of a tetrathreonine segment in hPrP that
clearly differs from the capped helices of chPrP and tPrP, and the
kinked helix of xlPrP (Figs. 1 and 3). An additional ‘‘structural
signature’’ for chPrP is provided by an insertion between the
helices 2 and 3 (Figs. 1 and 2), which forms a flexibly
disordered loop and an N-terminal elongation of helix 3 in
chPrP(121–225) (Fig. 3). This insertion is conserved in all known
avian PrP sequences (4).
Minimal Scaffold Maintaining the PrPC Architecture in Prion Proteins.
The structure comparisons in Fig. 3 indicate that the following
structure elements are preserved in all of the proteins of Fig. 2,
and form a scaffold that stabilizes the PrPC-type threedimensional
fold: The arrangement of the two long helices 2
and 3, their connection by a disulphide bond, the packing of the
-sheet against the helices 2 and 3 reinforced by a hydrogen
bond between T(S)183 O and Y162 HN, and the anchoring of
the helix 1 against the protein core by two tyrosine or tryptophane
residues in positions 149 and 150. Fig. 4 affords a
visualization of this preserved scaffold of PrPC, which illustrates
the variable orientation of helix 1 and indicates that variation
of the C-terminal extension of helix 2 by about two turns (Fig.
1) can be accommodated in a ‘‘PrPC-fold.’’
Indication for Function-Related Sequence Conservation in the Globular
Domain of PrPC. Most of the highly conserved amino acids in
the prion proteins shown in Fig. 1 have readily apparent structural
roles in the PrPC fold (see legend to Fig. 2), but there are
highly conserved residues in the peptide segment 146–153 of the
helix 1 (Fig. 1) that could, because of their peripheral location,
hardly be considered as structure-stabilizing elements (Fig. 5).
The structural motif of residues 146–153 was therefore used to
search a database of known protein sequences for functional
sites. A search of three-dimensional structures gave several hits,
which were exclusively helix–helix contacts within single domains.
A more extensive search that included sequences for
which no three-dimensional structures are available gave 100
hits, which eventually led to the indication of a significant
relationship between PrP segment 146–153 and region 351–358
of the human laminin 2 chain, which has the sequence EECYYDEN.
Because the laminin receptor precursor has been
repeatedly suggested as a possible protein partner for PrPC
(43–45), this indication of functional relations between the
laminin 2 chain and part of the helix 1 in PrPC might be worth
further study.
We thank Dr. T. Simonic for providing the turtle PrP plasmid, Dr. D.
Harris for providing the chicken PrP plasmid, and Dr. J. Gurdon
(University of Cambridge, Cambridge, U.K.) for the Xenopus laevis
cDNA library. Financial support was obtained from the Schweizerischer
Nationalfonds and the Eidgeno¨ssische Technische Hochschule (Zu¨rich)
through the National Center of Competence in Research Structural
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