The prion protein binds thiamine

The prion protein binds thiamineRolando Perez-Pineiro1,*, Trent C. Bjorndahl1,*, Mark V. Berjanskii2, David Hau1, Li Li3, Alan Hu-ang3, Rose Lee3, Ebrima Gibbs3, Carol Ladner1, Ying Wei Dong1, Ashenafi Abera1, Neil R. Cash-man3 and David S. Wishart1,2

1 Department of Biological Sciences, University of Alberta, Edmonton, AB, Canada

2 Department of Computing Science, University of Alberta, Edmonton, AB, Canada

3 Brain Research Centre, University of British Columbia, Vancouver, BC, Canada

Keywords

binding; in silico docking; NMR screening;

prion protein; thiamine

Correspondence

D. S. Wishart, Departments of Biological

Sciences and Computing Science, University

of Alberta, Rm. 2-21, Athabasca Hall,

University of Alberta, Edmonton, AB,

Canada T6G 2E8

Fax: +1 780 492 1071

Tel: +1 780 492 0383

E-mail: [email protected]

*These authors contributed equally to this

study

(Received 20 March 2011, revised 7 August

2011, accepted 11 August 2011)

doi:10.1111/j.1742-4658.2011.08304.x

Although highly conserved throughout evolution, the exact biological

function of the prion protein is still unclear. In an effort to identify the

potential biological functions of the prion protein we conducted a small-

molecule screening assay using the Syrian hamster prion protein [shPrP(90–

232)]. The screen was performed using a library of 149 water-soluble

metabolites that are known to pass through the blood–brain barrier. Using

a combination of 1D NMR, fluorescence quenching and surface plasmon

resonance we identified thiamine (vitamin B1) as a specific prion ligand

with a binding constant of � 60 lM. Subsequent studies showed that this

interaction is evolutionarily conserved, with similar binding constants being

seen for mouse, hamster and human prions. Various protein construct

lengths, both with and without the unstructured N-terminal region in

the presence and absence of copper, were examined. This indicates that the

N-terminus has no influence on the protein’s ability to interact with

thiamine. In addition to thiamine, the more biologically abundant forms of

vitamin B1 (thiamine monophosphate and thiamine diphosphate) were also

found to bind the prion protein with similar affinity. Heteronuclear NMR

experiments were used to determine thiamine’s interaction site, which is

located between helix 1 and the preceding loop. These data, in conjunction

with computer-aided docking and molecular dynamics, were used to model

the thiamine-binding pharmacophore and a comparison with other thia-

mine binding proteins was performed to reveal the common features of

interaction.

Introduction

Prion proteins (PrP) are endogenous, highly conserved

membrane-anchored proteins that are particularly

abundant in the neuronal cells of vertebrates. The

mature form of the normal cellular isoform of the

prion protein PrPc is a � 200-residue glycoprotein that

is tethered to the cell surface via a glycosyl-phosphati-

dylinositol anchor at the C-terminus [1]. A b-rich, mis-

folded isoform of PrP (generically designated PrPsc) is

the major macromolecule present in preparations of

infectious prions. Prions are known to cause a variety

of fatal, transmissible and incurable neurodegenerative

diseases in both animals and humans. These include

scrapie in sheep [2], bovine spongiform encephalopathy

in cattle [3], chronic wasting disease in deer and elk

[4], as well as Kuru, Creutzfeldt–Jacob disease and

fatal familial insomnia in humans [3,5,6]. Prions cause

Abbreviations

huPrP, human prion protein; KA, association constant; KD, dissociation constant; moPrP, mouse prion protein; PrPc, cellular prion protein;

PrPsc, scrapie prion protein; shPrP, Syrian hasmster prion protein.

4002 FEBS Journal 278 (2011) 4002–4014 ª 2011 The Authors Journal compilation ª 2011 FEBS

disease by converting from a soluble, helix-rich form

(PrPC) to an infectious b-rich form (PrPsc) that is both

insoluble and highly pathogenic [7].

After more than 30 years of study very little is

known about the physiological role of prion protein.

Most of the studies published to date have focused on

the identification of PrP-interacting partners such as

Cu2+, Ni2+, glycosaminoglycans, DNA, RNA and a

number of signaling proteins [8]. Based on these obser-

vations, the proposed physiological roles of PrPc range

from copper internalization and homeostasis to a vari-

ety of anti-apoptotic activities. Other potential func-

tions include protection against oxidative stress, cell

adhesion, cell signaling and the modulation of synaptic

structure and function [9,10]. More recent findings

suggest that PrPc may play a role in maintaining the

long-term integrity of peripheral myelin sheaths [11] or

even function as an antibacterial protein [12].

Aside from the identification of Cu2+ (and other

divalent metals such as Ni2+, Zn2+, Fe2+ and possibly

Mn2+) and hemin [13] as high-affinity PrP ligands,

there has been no published evidence that PrPc binds

any other endogenous small molecules. That is not to

say that PrP isoforms cannot, or do not, bind small

molecules. Indeed there are numerous studies that have

identified a variety of exogenous or xenobiotic ligands

that bind to either PrPc or PrPsc with relatively high

affinity. These include tetracyclines [14], quinacrines

[15], curcumin [16], simvastatin [17], Congo Red [18]

and others. However, these small molecules are not

endogenous molecules and they were identified through

chemical screens aimed at finding potential prion

therapies rather than potential PrP functions.

In an effort to identify potentially physiologically rel-

evant binding partners of the prion protein, we investi-

gated the binding of small molecules that would be

easily accessible to prion proteins. Taking into account

the extracellular location and the enrichment of PrPc in

the central nervous system, we chose to look at a collec-

tion of water-soluble metabolites that could easily pass

through the blood–brain barrier and which are found in

high abundance in central nervous system tissues or

biofluids. More specifically, we decided to screen recom-

binant prion proteins against a subset of 149 water-sol-

uble metabolites that were previously identified as being

enriched in human cerebrospinal fluid [19]. Using a

combination of 1D NMR, fluorescence quenching and

surface plasmon resonance, we found that thiamine

(vitamin B1) was the only ligand that exhibited clear

and specific binding to PrPc. The binding constant was

determined to be � 60 lm. Subsequent assessment of

thiamine binding showed that this interaction is evolu-

tionarily conserved, with similar binding constants

being seen for hamster, mouse and human prion pro-

teins. We also assessed the binding of other, more phys-

iologically abundant thiamine derivatives (thiamine

monophosphate and thiamine diphosphate) and deter-

mined the exact binding site for thiamine using a com-

bination of 2D heteronuclear NMR experiments and

computer-aided docking and molecular dynamics.

Results

1D NMR screening

One-dimensional NOESY NMR screening was per-

formed on a total of 149 water-soluble metabolites

(Table S1) in the presence and absence of recombinant

Syrian hamster prion protein [shPrP(90–232)]. Spectra

were compared for chemical shift, linewidth and ⁄orlineshape perturbations of the metabolite proton sig-

nals. The criteria for selecting potential binders

included the presence of new proton chemical shifts or

perturbations > 0.02 ppm, > 10% reduction in peak

intensity and ⁄or > 0.2 Hz broadening of the metabo-

lite signals. The parameters for the compound only

and compound + protein spectral collection were opti-

mized for detecting small molecules. Through this

NMR analysis, we identified two potential binders that

fit our criteria: thiamine and cytidine (Fig. 1A).

SPR and fluorescence studies of the

thiamine-prion complex

The binding of the two candidates identified from our

1D NMR screen to the prion protein, (Fig. 1A) were

also characterized using SPR as a second, independent

method. At ligand concentrations of 10 mm, binding is

clearly observed for thiamine (Fig. 1B). By contrast, the

affinity for cytidine to PrP as measured by SPR was

found to be very weak and we decided not pursue

further studies with this ligand. Caffeine, a negative con-

trol, exhibited nonspecific binding as manifested by the

negative binding response after dissociation. As shown

in Fig. 1C, the biosensor response arising from thiamine

binding is concentration dependent. Values of the asso-

ciation and dissociation constant (KD = 116 · 10)6m)

for this ligand were calculated using regression analysis

of the binding data.

Steady-state fluorescence quenching is a valuable

technique to study ligand–protein interactions if the

ligand binds near tyrosine or tryptophan residues [20].

Assessment of thiamine binding to PrP using

fluorescence quenching indicated a decrease in the flu-

orescence signal of shPrP at both 295 and 280 nm

after addition of thiamine (Fig. 2A) and the calculated

R. Perez-Pineiro et al. PrP binds thiamine

FEBS Journal 278 (2011) 4002–4014 ª 2011 The Authors Journal compilation ª 2011 FEBS 4003

Stern–Volmer plot was linear for the selected concen-

tration range. Using the fluorescence data (see Materi-

als and methods) the estimated values for the

disassociation constants (KD = 65.36 · 10)6m) were

determined (Table 1, row 1). Interestingly, a similar

quenching profile was found for a longer version of

the Syrian hamster prion protein, shPrP(29–232) with

a histidine affinity tag (Table 1, row 2), the mouse

prion protein, moPrP(90–231) (Table 1, row 3) and

the full-length human prion protein, huPrP(23–230)

with histidine affinity tag (Table 1, row 4). These

results indicate that the binding of thiamine is con-

served across a wide range of mammalian species. Fur-

thermore, they also show that the binding is not

Fig. 1. (A) Chemical structures of thiamine and cytidine. (B) Biosen-

sor analysis of binding of thiamine, caffeine (negative control) and

cytidine to shPrP (90-232). (C) Biosensor response due to the

binding of thiamine (0 lM – light red, 50 lM – light magenta,

100 lM – yellow, 200 lM – cyan, 400 lM – blue, 800 lM – dark red,

1.6 M – dark magenta, 3.2 M – green) to the protein is concentra-

tion-dependent.

Fig. 2. (A) Fluorescence emission spectra of shPrP(90–232), 20 lM

in the presence of thiamine at different concentrations, (1) 0 M, (2)

10 lM (3) 20 lM (4) 40 lM (5) 80 lM (6) 160 lM (7) 250 lM (8)

320 lM (9) 510 lM (10) 700 lM; k = 295 nm. (B) The Stern–Volmer

plot of fluorescence quenching of shPrP(90–232) by thiamine.

Values for KD (65.36 · 10)6M), were obtained according to

equation (1).

PrP binds thiamine R. Perez-Pineiro et al.

4004 FEBS Journal 278 (2011) 4002–4014 ª 2011 The Authors Journal compilation ª 2011 FEBS

affected by the presence of the His-tag or the unstruc-

tured, copper-binding N-terminus. Similar fluorescence

quenching of shPrP(29–232) was observed for the

mono and diphosphate analogs of thiamine and for

the moPrP(90–232) in the presence of a 3 m excess of

CuCl2. The calculated KA and KD values for these

constructs were in the same range of nonphosphorylat-

ed thiamine with the C-terminal core of the shPrP(90–

232) protein (Table 1, rows 5–9). The Stern–Volmer

plots are shown in Figs S1–S5.

Thiamine binding

Additional NMR studies were conducted to further

validate the PrP–thiamine binding and to identify the

site of interaction. A saturation transfer difference

TOCSY spectrum (STD-TOCSY) was collected to

identify the ligand protons that interacted with the

protein [21]. As observed from the reference spectrum

(Fig. S8B), the majority of thiamine protons, apart

from the methyl groups and the methylene signal at

5.3 p.p.m., were completely suppressed. Upon addition

of the shPrP(90–232), recovery of the remaining signals

was observed (Fig. S8C), whereas the methyl groups

and methylene proton displayed significant signal

Table 1. Binding affinity (KD) and correlation coefficients (C ) of

thiamine for different prion protein (PrP) constructs according to

equation (1).

Entry Ligand PrP construct KD (M) C

1 Thiamine shPrP(90–232) 65.36 · 10)6 0.9892

2 Thiamine shPrP(29–232) 66.66 · 10)6 0.9811

3 Thiamine moPrP(90–231)a 58.82 · 10)6 0.9679

4 Thiamine huPrP(23–230) 62.11 · 10)6 0.9693

5 Thiamine

(PO4)

shPrP(29–232) 72.72 · 10)6 0.99

6 Thiamine

(PO4)bshPrP(29–232) 69.52 · 10)6 0.9810

7 Thiamine +

CuCl2c

moPrP(90–232) 56.82 · 10)6 0.9991

8 Thiamine

(PO4) + CuCl2c

moPrP(90–232) 65.79 · 10)6 0.99

9 Thiamine(PO4)2 +

CuCl2c

moPrP(90–232) 59.88 · 10)6 0.99

10 Thiamine ShPrP(90–232)

pH 6.0

67.71 · 10)6 0.99

11 Thiamine ShPrP(90–232)

pH 8.0

68.90 · 10)6 0.99

a No His-tag. b Value calculated based on tyrosine quenching.c Three molar excess (3 ·) of CuCl2 in relation to the protein and

ligands were used.

10 9 8 7

H1 p.p.m.

130129128127126125124123122121120119118117116115114113112111110109108107

N15

p.p

.m.

10 9 8 7131

Fig. 3. Overlapped 2D 15N-HSQC spectra of shPrP(90–231) 500 lM in 20 mM K2HPO4 (pH 6.2) without thiamine (black) and with a 33 molar

excess of thiamine HCl (red). Residues displaying significant signal attenuation upon addition of thiamine are annotated, including D144,

G142, F141 and M138. Those residues found to be in direct contact with thiamine are annotated in dark red (large font size), whereas those

experiencing distal ligand-induced conformational effects are annotated blue and yellow (smaller font size). A ribbon representation of

shPrP(90–232) with bound thiamine is shown in the inset.

R. Perez-Pineiro et al. PrP binds thiamine

FEBS Journal 278 (2011) 4002–4014 ª 2011 The Authors Journal compilation ª 2011 FEBS 4005

enhancement, indicating that all of thiamine’s protons

are involved in interaction with the prion protein.

The thiamine binding site was investigated using

heteronuclear NMR spectroscopy on a 15N-labeled

sample of the shPrP(90–232 with 6 · -His tag) protein

construct. Figure 3 shows the signal attenuation and

chemical shift changes in the 15N-HSQC spectrum of

the shPrP after addition of thiamine (33 : 1,

ligand ⁄protein molar ratio). The majority of attenu-

ated signals (M138, F141, G142 and D144) and those

with significant chemical shift perturbations (H140,

N143 side chain) cluster in an unstructured loop

adjacent to helix 1, which contains two tyrosines and a

tryptophan residue. These aromatic residues are within

the required 10 A of the interacting ligand to exhibit

fluorescence quenching at both 280 nm (Tyr, Trp exci-

tation) and 295 nm (Trp excitation). The perturbation

of the N143 side-chain amide is also noteworthy.

Although it is not in direct contact with thiamine, its

binding results in slight changes of the hydrogen bond

length between it and the E146 side-chain carboxylic

acid group. In the majority of the NMR structures

(PDB: 1B10), these side chains are involved with the

N-terminal capping of helix 1. In addition, a second

region of the protein exhibits conformation change,

which is likely a consequence of small changes in

hydrogen bond lengths or other minor conforma-

tional ⁄dynamic changes. However, no visible NOEs

could be observed between thiamine and this region,

which consists of the ‘amylome’ residues F169-

NNQNNY-175. Long-range effects between this region

and the distal residues in helix 3 have been noted in

previous studies [22]. Whether these distal effects on

the amylome region upon thiamine binding have any

significance or are an inconsequential artifact has yet

to be determined.

Although 15N-HSQC titration spectra can be useful

in identifying residues directly perturbed by ligand

binding, they are also sensitive to small perturbations in

hydrogen bond length, solvent structure and electric

field effects not directly related to ligand binding, result-

ing in a number of false-positive signals. Evidence of

this can be seen in the HSQC data presented in Fig. 3.

Also shown are a number of resonances that exhibited

some signal attenuation, likely due to ligand-induced

changes in the protein’s dynamics (i.e. intermediate

conformational exchange). In light of these hard-to-

interpret signal perturbations, we felt that additional

data were necessary to corroboate the initial findings.

Consequently, a NOESY experiment (Varian VNMRJ

v2.1b: tnnoesy) was performed on a sample containing

a 50 : 1 molar ratio of thiamine to the shPrP(90–232).

This experiment provided five unique NOE signals that

could be clearly assigned to protons within the putative

binding pocket (Fig. 4). Confirmation of the proton

chemical shift assignments was made from 15N TOC-

SY-HSQC and NOESY-HSQC data. Strip plots from

the residues providing NOE data are also provided in

the supporting information (Fig. S7). The attenuated

amide signal for G142 in the 15N-HSQC spectrum

tracks from 9.05 to 8.90 p.p.m. upon addition of

thiamine. This perturbation is also observed in the

TOCSY-HSQC data (Fig. S7), which dislays both free

and bound states in slow conformational exchange. The

Fig. 4. Regions of the tnnoesy spectrum collected on a 0.5 mM

shPrP(90–232) sample (20 mM KH2PO4, pH 7.0) with a 50-fold

molar excess of thiamine showing (A) the thiazolium proton

(9.38 p.p.m.) and the pyrimidine proton (8.00 p.p.m.) and (B) the

downfield shifted alkyl proton nearest to thiamine’s hydroxyl group.

The NOEs used to select the docking candiate shown in Fig. 5 are

annotated. Intrathiamine NOEs present a negative intensity (red),

while intermolecular shPrP to thiamine NOEs are positive (black).

Chemical shift values are shown and correspond to those identified

assigned in 15N-edited TOCSY-HSQC and NOESY-HSQC experi-

ments collected at pH 7.0 (Fig. S7A, B).

PrP binds thiamine R. Perez-Pineiro et al.

4006 FEBS Journal 278 (2011) 4002–4014 ª 2011 The Authors Journal compilation ª 2011 FEBS

tnnoesy spectum exhibits a very strong NOE between

thiamine’s aliphatic proton shift at 3.83 p.p.m. and

G142 amide proton (at 8.90 p.p.m.) in the bound state

(Fig. 4B). Other strong NOE data were observed

between thiamine’s pyrimidine and isoxazole ring pro-

tons and the protons for residues M138, M139, H140

and Y150 aromatic rings (Fig. 4A). From these NMR

data, the orientation of thiamine in complex with PrP

was modelled in-silico (Fig. 5). Analysis of the docking

result revealed that it was in good agreement with the

observed NMR and fluorescence quenching data. Spe-

cifically in this model, all NOEs were satisfied, the

ligand made good contact with residues displaying

NMR perturbations and the placement of the ligand is

within 10 A of W145, Y149 and Y150.

Discussion

By analyzing the fluorescence data, some additional

clues about the nature of PrP–thiamine interaction

could be obtained. The linearity of the Stern–Volmer

plot proves there is only one fluorogen that interacts

with the quencher (thiamine). However, these data alone

cannot distinguish between a dynamic or a static

quenching mechanism. To clarify this issue, we observed

only a slight decrease in the slope of the Stern–Volmer

plot when the experiments were conducted at higher

temperatures (40 �C, data not shown). This result is

characteristic of static quenching. By using equation (1):

Fo=F ¼ 1þ Kqs0½Q� andKA ¼ 1=KD ¼ Kqs0 ð1Þ

It is possible to calculate the apparent quenching con-

stants Kq of the two metabolites assuming the lifetime s0

of the biomolecules to be � 10 ns. It is known that the

maximal diffusion collision quenching constant of

quenchers is 2.0 · 1010 mÆs)1 [23]. In this case, the

apparent quenching constant calculated for thiamine

(1.53 · 1012 mÆs)1) is larger than the diffusion-con-

trolled quenching constant suggesting that the quench-

ing effect of thiamine on the prion protein is not due to

dynamic collisions. This result supports the fact that the

origin of the observed quenching effect is due to the for-

mation of a ground-state complex between the protein

and thiamine. We have also excluded a Forster energy

transfer mechanism as the other probable cause of static

quenching because in this case the fluorescence emission

spectrum of the prion protein does not overlap with the

UV absorption spectrum of the thiamine.

The results obtained from our SPR studies con-

firmed the thiamine affinity measurements made by flu-

orescence (� 65 lm). However, the KD value obtained

from SPR (� 116 lm) is about half that obtained by

our fluorescence experiments. The lower binding con-

stant as measured from SPR was not unexpected.

Indeed, given the fact that the prion protein was cova-

lently coupled (via lysine and arginine residues) to the

SPR chip, one would expect that this chemical cross-

linking would lead to a portion of the PrP molecules

having their binding sites shielded or inaccessible. It is

also notable that three arginine residues surround the

putative binding site (R136, R148 and R151). Further-

more, reduced binding constants are expected given

that some denaturation of the native structure is often

induced by the covalent modification [24].

As noted earlier, the prion protein does bind to other

endogenous ligands, including copper, zinc, manganese

and nickel. The binding sites for these metals are located

between residues 29 and 98, in the unstructured, N-ter-

minal octarepeat region [25,26], well away from where

thiamine binds. In most cases, the metal binding con-

stants are quite strong. The KD for copper is an aston-

ishingly tight 8 fm, the KD for nickel is � 20 nm, the KD

for zinc is � 100 nm, and the KD for manganese is rela-

tively weak at 200 lm [25]. The other endogenous ligand

that appears to bind to PrP is hemin, a naturally occur-

ring iron-binding porphyrin. Hemin appears to bind

exclusively in the unstructured N-terminus from resi-

dues 34 to 94, which corresponds to the octarepeat

region [13]. However, the binding affinity of hemin has

not yet been determined. Some non-natural porphyrin

analogs of have also been shown to bind to PrP with

low micromolar affinity [27] so it is likely that the KD

value for hemin would be in the same range. Among

nonendogenous or xenobiotic compounds that have

been shown to bind the cellular form of PrP, only quina-

crine has had both its binding site and binding affinity

Fig. 5. Thiamine docked onto a ribbon representation of the

shPrP(90–232). Residues displaying 15N-HSQC signal attenuation

are colored red. The NOEs obtained from the transferred NOE

experiment (supporting information) used to generate the model

are indicated with dashed yellow lines. The ribbon is colored yellow

at positions W145, Y149 and Y150, which are the residues respon-

sible for the fluorescence quenching results.

R. Perez-Pineiro et al. PrP binds thiamine

FEBS Journal 278 (2011) 4002–4014 ª 2011 The Authors Journal compilation ª 2011 FEBS 4007

determined. In particular, quinacrine binds to the C-ter-

minal region of helix 3 with a rather low KD of 4.3 mm

[15]. Overall, these data suggest that the affinity of the

prion protein for thiamine is within the range of other

known PrP ligands. Furthermore, the thiamine binding

site appears to be unique and, so far as we know, does

not overlap with the binding site of other known

(endogenous or exogenous) prion ligands.

To assess whether the binding of thiamine to PrP

might share features with other thiamine binding pro-

teins, we compared our PrP–thiamine structure with

other known thiamine binding proteins including:

transketolase, thiamine pyrophosphokinase (from

mouse and Bacillus subtilus), thiamine phosphate syn-

thase and thiamine binding protein (Fig. 6). For all the

thiamine binding proteins, the amino substituted

pyrimidine ring occupies a hydrophobic pocket. Stabil-

ization and orientation of the ring is further provided

through hydrogen bond acceptor oxygen atoms. In the

case of pyrophosphokinase, salt bridges are also found

to the positively charged imidazolium nitrogen.

Finally, electrostatic and hydrogen bonds from the

backbone amides and side chain amide (Q, N) or

hydroxyl groups (S, T) and charged residues (D, E, R,

K and H) provide contacts for the thiamine hydroxyl

group or various phosphorylated forms. For the prion

protein, similar contacts are observed. Specifically, the

pyrimidine ring of thiamine sits in a hydrophobic

pocket created by Y150, Y141 and M138, and is also

stabilized electrostatically by the backbone carbonyl of

M139 and the Y150 hydroxyl group. In addition, the

positively charged imidazolium nitrogen forms a salt

bridge with the carboxylic acid side chain from D147,

and finally, the thiamine hydroxyl group is located

within a hydrogen-bond distance range (2.5 A) from

the backbone amide proton of G142. There are a num-

ber of additional residues (N143, D144, D147, R148)

in this region that can offer either electrostatic or

hydrogen bonding interactions to either the thiamine

hydroxyl group or its phosphate groups. Interestingly,

Fig. 6. Pharmacophore comparison between shPrP(90–232) and other thiamine binding proteins. The modeled structure of the shPrP(90–

232) protein with docked thiamine is shown in (A). (B) Thiamine phosphate synthase (PDB: 2TPS), (C) mouse thiamine pyrophosphokinase

(PDB: 1IG3), (D) Bacillus subtilis thiamine pyrophosphokinase (PDB: 3LM8), (E) thiamine binding protein (PDB: 2QRY) and (F) transketolase

(PDB: 3M34).

PrP binds thiamine R. Perez-Pineiro et al.

4008 FEBS Journal 278 (2011) 4002–4014 ª 2011 The Authors Journal compilation ª 2011 FEBS

it was noted that the H140 side chain played no direct

part in the interaction of thiamine. In order to validate

this observation, we performed additional binding

studies at pH 6 and 8, thereby titrating H140 through

its full range of pKa values [28], as well as most known

physiological pH ranges. The fluorescence quenching

data yielded no difference in the calculated KD con-

stants for these pH values, indicating that pH does not

influence thiamine binding (Table 1, Fig. S6). Other

prion protein constructs from nonmammalian species

(turtle and frog) show variability in the amino acid at

this position (N and Q respectively). These findings

suggest that the amino acid in this position plays no

significant role in the protein affinity for thiamine.

Potential biological consequences

Thiamine (vitamin B1) is an essential, water-soluble,

B vitamin that plays a critical role in carbohydrate

metabolism [29]. It is endogenously synthesized by bac-

teria and plants, but animals cannot synthesize it, so

thiamine must be obtained from the diet. Generally,

the unphosphorylated form is transported in the body,

whereas the phosphorylated forms of thiamine

(thiamine monophosphate, thiamine diphosphate and

thiamine triphosphate) are the active forms of the vita-

min. The human body keeps stores of 25–30 mg of

thiamine, with the greatest concentrations being in

metabolically active organs, such as skeletal muscle,

the brain, the heart, the kidneys and the liver [30].

Thiamine is known to bind to serum albumin [31], to a

hormonally regulated protein called thiamine binding

protein [32] and to a thiamine ⁄ folate transporter [33].

Thiamine binding protein and serum albumin have an

affinity for thiamine of � 1 lm [31,32]. Thiamine and

its phosphorylated derivatives have also been detected

in blood, cerebrospinal fluid, milk and several other

biofluids. The typical concentration of all forms of thi-

amine (free and phosphorylated) in human blood is

� 200–300 nm [30]. Thiamine is absorbed by active

transport and by passive diffusion.

Given the relatively low concentrations of thiamine

in the body, one might ask how a protein with a mod-

est (� 60 lm) affinity to thiamine could potentially

play a biologically meaningful role. One possibility is

that the prion protein functions to retain or concen-

trate thiamine in tissues more through avidity rather

than affinity. Many copies of a weak binding protein

on a cellular surface can create a ‘Velcro’ effect for

ligand binding. It has been estimated that prion pro-

teins are expressed at a level of between 2000 and 4000

copies per cell in peripheral tissues and organs and as

much as 50 000 copies per cell in cerebral tissue [34].

Clearly, if the role of PrP is to concentrate thiamine in

tissues, it would need to have a relatively weak binding

constant so that the thiamine could be released for

absorption and subsequent utilization by cells. It is

also important to note that the thiamine binding

constant reported here was determined for a soluble,

unglycosylated form of PrP rather than the native,

membrane-bound form. It may be that native, mem-

brane-bound PrP could exhibit higher affinities for thi-

amine because of the presence of the lipid bilayer or

other synergistic protein–protein interactions. It is

intriguing that prion proteins are highly expressed in

the brain, spinal cord, heart, kidney, lung, white blood

cells and lymphoid tissues [35]. These correlate well

with the tissues typically needing the highest levels of

thiamine in the body and the tissues with the highest

levels of carbohydrate metabolism.

In conclusion, we have identified, from an initial

screening of 149 water-soluble metabolites commonly

found in cerebrospinal fluid that the water-soluble vita-

min B1, thiamine, interacts with multiple constructs of

the prion protein. Three independent methods were

used to confirm the interaction. Two of them (SPR and

fluorescence quenching) led to binding constants in the

weak lm range, and NMR studies pinpointed the site

of interaction. Further docking studies were used to

ascertain the hydrogen bonds, electrostatic and lypo-

philic interactions that comprise the pharmacophore.

The residues involved with these interactions are

conserved across multiple mammalian species (Fig. 7).

Additional experiments with mouse, human and vari-

ous hamster prion constructs showed that this binding

was conserved across these mammalian species.

Materials and methods

Protein expression and purification

The expression and purification of recombinant shPrP(29–

232), shPrP(90–231) and shPrP(120–232), moPrP(90–231)

and huPrP(23–230) all followed a similar protocol.

Fig. 7. Structural sequence alignment for various prion protein con-

structs over the region containing the residues displaying 15N-HSQC

signal attenuation upon the addition of thiamine. The structure

alignment was performed using PYMOL (Warren Delano, ª 2004).

Residues showing phamacophore interactions with thiamine are

bolded italics. Hyrophobic residues are colored yellow, acid residues

are colored red, basic residues are colored blue.

R. Perez-Pineiro et al. PrP binds thiamine

FEBS Journal 278 (2011) 4002–4014 ª 2011 The Authors Journal compilation ª 2011 FEBS 4009

Specifically, synthetic genes corresponding each construct

including a 22-residue N-terminal fusion tag containing

6 · His and a thrombin cleavage site (MGSSHHHHHH

SSGLVPRGSHML) were synthesized by DNA 2.0

(Menlo Park, CA, USA). The genes were cloned into a

pET15b expression vector between XhoI and EcoRI

restriction sites and heat shock transformed into Escheri-

chia coli strain BL21 (DE3). For expression, the trans-

formed cells were grown in 100 mL Luria–Bertani broth

plus 100 lgÆmL)1 ampicillin overnight to generate a star-

ter culture. Between 1% and 2% of this starter culture

was then used to inoculate 1 L of Luria–Bertani media

(giving a starting D600 of 0.1). The cells were allowed to

reach an D600 between 0.6 and 1.0 before induction with

1 mm isopropyl thiogalactoside. Twelve to eighteen hours

later, the cells were harvested by centrifugation at 1600 g

for 25 min at 4 �C. In addition, 15N-labeled shPrP(90–

232) was also expressed and purified from M9 media

(1.0 gÆL)1 15NH4Cl) for collection of heteronuclear NMR

data. The inclusion of the 6· His tag afforded a stan-

dardized nickel affinity purification strategy for all pro-

tein constructs. The details of the purification protocol

are described elsewhere [36].

NMR experiments

NMR spectra were acquired at 25 �C on a 500 MHz

Varian Unity INOVA spectrometer fitted with a 5 mm

HCN z-gradient pulsed-field gradient cryogenic probe

except for the STD-TOCSY, which was collected at 25 �Con a 800 MHz Varian Unity INOVA spectrometer fitted

with a 5 mm HCN xyz-gradient pulsed-field gradient cryo-

genic probe. All experiments were collected using Varian

BioPack pulse sequences (VNMRJ v2.1B). Spectra were

processed using nmrpipe [37] and analyzed with nmrpipe

and nmrviewj [38] unless stated otherwise.

Small molecule metabolites used in the ligand screening

were purchased from Sigma-Aldrich (St. Louis, MI, USA),

Fisher Scientific (Waltham, MA, USA), Acros Organics

(Geel, Belgium) and Alfa Aesar (Ward Hill, MA, USA)

and used without further purification. The final (cerebrospi-

nal fluid-compatible) metabolite library consisted of 149

different water-soluble chemicals. The complete list is

available in the Table S1. Seventeen screening sets contain-

ing between six and nine metabolites were manually

selected with the aid of Chemaxon’s jklustor v 5.0

(ChemAxon Kft, Budapest, Hungary) using the Ward algo-

rithm. Selection of each chemical set was made on the basis

of minimizing 1H NMR spectral overlap. Two sets of

1D NOESY spectra were collected on each set of metabo-

lites (with and without PrP protein). To collect the first

NOESY NMR spectrum, each metabolite set was dissolved

in 20 mm potassium phosphate buffer at pH 6.5 giving a

final concentration of 100 lm. Ten percent (v ⁄ v) D2O was

added to each sample to maintain a spectral lock, 1 mm of

2,2-dimethyl-2-silapentane-5-sulfonate was added for chemi-

cal shift referencing [39]. For each 1D NOESY spectrum,

48 000 points were averaged from 256 transients over a

sweep width of 6000 Hz. A recycle delay of 0.01 s and an

acquisition time of 4 s were used. The mixing time for the

screening experiments was 100 ms. Immediately following

collection of the reference compound spectra, the NMR

sample was used to reconstitute prealiquoted, lyophilized

shPrP(90–232) and a second spectrum was collected with

the same parameters. The molar ratio of protein to each

compound was 1 : 1. Matching spectra were superposed

and analyzed with the Chenomx NMR suite v6.0 to assess

chemical shift and linewidth perturbations of the metabolite

signals.

Saturation transfer difference TOCSY experiments were

collected on a 12.5 mm sample of thiamine (20 mm

K2HPO4, pH 7.5, 10% D2O) before and after the addition

of 500 lm shPrP(90–232). The spectra were collected using

4096 transients with a sweepwidth of 12000 Hz, a mixing

time of 10 ms, a recycle delay of 1 s and an acquisition

time of 2 s. On and off irradiation frequencies corre-

sponded to )0.73 and 24.5 p.p.m. respectively.

To collect the 2D 15N-HSQC titration spectra, a reference15N-HSQC spectrum of the shPrP(90–232), alone, was first

collected (300 lm, 350 lL, 20 mm KH2PO4, pH 7 and 6.2).

These 15N-HSQC reference spectrum was collected with

2048 complex points in the 1H dimension and 256 complex

points in the 15N dimension using a recycle delay of 1.5 s

(nt = 120, sw = 6000 Hz, sw1 = 1800 Hz). Thiamine HCl

was successively added to concentrations of 1, 2, 5 and

10 mm and the 15N-HSQC spectra recollected using identi-

cal acquisition parameters. The amide chemical shift data

for the prion-thiamine complex has been deposited into the

BioMagResBank (BMR17834).

A 2D tnnoesy experiment (Varian VNMRJ v2.1b) was

collected on a 500 lm shPrP(90–232) sample with 25 mm

thiamine in 20 mm KH2PO4, (pH 7.0, 350 lL, 10% D2O)

at 25 �C. The mixing time was 50 ms and a 1.5 s recycle

delay was used. Sixty-four transients were collected with

sweep widths of 6000 Hz in both the direct and indirect

detected dimensions (np = 1024, ni = 128). Three-dimen-

sional 15N edited TOCSY-HSQC and NOESY-HSQC [40]

experiments were acquired with a 500 lm shPrP(90–232)

sample prepared with 10 mm thiamine in 20 mm KH2PO4

(pH 7.0, 10% D2O, 350 lL). Sixteen transients were col-

lected for each experiment with sweepwidths of 6000 Hz

in the direct and first indirectly detected dimensions

(np = 1024, ni = 64). A sweep with of 1800 Hz was

used for the second indirectly detected dimension

(ni2 = 32). Mixing times of 50 and 100 ms were used for

the TOCSY and NOESY experiments respectively. A

recycle delay of 1.5 s was used and the experiments were

collected at 25 �C. All samples were transferred to

Shigemi (Shigemi Inc. Allison Park, PA, USA) microcell

NMR tubes (350 lL) prior to spectral acquisition.

PrP binds thiamine R. Perez-Pineiro et al.

4010 FEBS Journal 278 (2011) 4002–4014 ª 2011 The Authors Journal compilation ª 2011 FEBS

Steady-state fluorescence quenching

measurements

Fluorescence emission spectra were recorded on a PTI

MODEL-MP1 spectrofluorometer using a 1 cm fluores-

cence cell for all measurements. Two different excitation

wavelengths (295 and 280 nm) were used and the scan

range was 310–450 nm. Prior to collecting the fluorescence

spectra, the prion protein (20 lm) was dissolved in 100 lLof 20 mm potassium phosphate buffer at pH 6.0, 7.0 or 8.0

and incubated with increasing concentrations of the metab-

olites of interest (10–700 lm) for 30 min with shaking (800

rpm). To analyze the effect of copper on the binding of thi-

amine and thiamine-phosphate analogs to PrP, CuCl2 was

added to the mixture in a threefold excess previous to the

addition of the ligand. Data from these fluorescence experi-

ments were used to determine the apparent binding con-

stant according to equation (2):

Fo=F ¼ 1þ KA½Q� ð2Þ

Where KS = KA, is the formation constant of the donor–

acceptor (quencher–fluorogen) complex. The concentration

of the quencher [Q] after titration is taken to be its ratio to

protein concentration [Pt]: [Q] ⁄ [Pt]. From the slope of the

linear plot of Fo ⁄F versus [Q] ⁄ [Pt] the binding constant and

dissociation constant (1 ⁄KA) were estimated. The results are

expressed as mean values ± SD (n = 5–7).

SPR measurements

The SPR data was collected on a Biacore� system 3000

(BIAcore, Uppsala, Sweden) equipped with a CM5 sensor

chip. HBS-EP buffer (10 mm Hepes pH 7.5, 150 mm NaCl,

3.4 mm EDTA, 0.005% surfactant P20) was used for immo-

bilization of the protein. CM-dextran on A CM5 sensor chip

was activated by mixing equal volumes of 50 mm N-hydroxy

succinimide and 200 mm 1-ethyl-3-(3-dimethylaminopropyl)

carbodiimide followed by injection of the mixture over the

sensor chip surface for 7 min at a flow rate of 5 lLÆmin)1.

The shPrP(90–232) to be immobilized was injected over the

surface for 7 min. The unreacted sites on the sensor chip sur-

face were blocked by injection of 1 m ethanolamine, pH 8.5

for 7 min. Thiamine was diluted in HBS-EP-0.05% and

simultaneously injected over the PrP flow cell and the refer-

ence for 3 min at a flow rate of 30 lLÆmin)1. The dissocia-

tion phase was monitored for 2.5 min. The flow cell was

washed with glycine ⁄HCl (pH 1.7) at 60 lLÆmin)1 for 30 s

between each sample injection. Included in the assay were

positive controls (quinacrine HCl and congo red), com-

pounds known to bind shPrP(90–232), and a negative con-

trol, caffeine. The resultant sensorgrams, a plot of binding

response over time, were all double-referenced by first sub-

tracting the binding to the reference surface from the binding

to the active surface, and further subtracting out the binding

response of the sample diluents from all sensorgrams. The

dissociation constant (KD) for thiamine HCl was calculated

from data at doses ranging from 50 lm to 3.2 mm. Dilution

series sensorgrams were then evaluated using the steady-state

affinity analysis protocol of the biaevaluation 4.1.1 soft-

ware (GE HealthCare, Piscataway, NJ, USA), to obtain

affinity constants.

In silico docking protocol

The in silico docking protocol was initiated by screening

models of the shPrP NMR ensemble, PDB: 1B10 [41], for

structures having the propensity to bind thiamine in orien-

tations that are consistent with the NMR data. This step

was carried out using autodock v4.2 [42]. Coordinates of

the thiamine molecule in PDB format were obtained from

the Human Metabolome Database [18]. Hydrogen atoms

were added to the thiamine molecule using the openbabel

program [43]. Non-polar hydrogens in PrP and thiamine

were identified and merged with heavy atoms by auto-

dock tools 1.5.4 [44]. autodock tools were also

employed for calculating Gasteiger charges for both the

thiamine and PrP models. Six rotatable torsion angles in

thiamine molecule were identified by autodock tools and

allowed to freely rotate to perform flexible docking. The

protein model was treated as a rigid body during the

docking simulations. A large grid box (25 · 25 · 30 A)

centered in the vicinity of NMR-mapped thiamine binding

site (residues M138, M139, H140, F141, G142, D144 and

W145) was generated. This box also included helix 1, the

loop between b-sheet 1 and helix 1, the C-terminal half of

helix 2, the loop between helices 1 and 3, and the N-termi-

nal half of helix 3. Grid spacing was set to 0.375 A. The

atom-specific affinity map, electrostatic potential map, and

desolvation potential map were generated by the auto-

grid 4 program from the autodock package. The initial

dihedral offset of the ligand and the initial position of

ligand with respect to the protein model were selected ran-

domly for every docking run. A Lamarckian genetic algo-

rithm was selected to perform the docking simulations.

Two hundred and fifty docking runs were conducted using

the default autodock 4.2 parameters, except the following:

250 individuals in the ligand population, 2 500 000 energy

evaluations, 0.2 A translation step, 5� quaternion step, 5�torsion step.

During the second step, semiflexible docking was per-

formed with xplor-nih 2.27 [45] on the shPrP NMR

structure (PDB: 1B10 :model 13). This model showed

good agreement with the NOE data within the 6 A

upper-limit in our autodock simulations. Ligand and

protein side-chains were allowed to be flexible, while pro-

tein backbone was kept rigid. Thiamine topology and

parameter files for xplor were generated with the acpype

program (http://code.google.com/p/acpype/). After a

short initial energy minimization (50 steps), the thiamine

R. Perez-Pineiro et al. PrP binds thiamine

FEBS Journal 278 (2011) 4002–4014 ª 2011 The Authors Journal compilation ª 2011 FEBS 4011

orientation was optimized with Cartesian dynamics for

6000 constant-temperature steps (T = 1000 K) and 6000

steps of simulated annealing to the final temperature of

300 K, followed by 400 minimization steps. A model that

had no NOE violations was selected as the final docking

pose. The coordinate data for this model has been depos-

ited into the Protein Data Bank (PDB accession no.

2LH8).

Acknowledgements

This project was funded by PrioNet Canada, the

Alberta Prion Research Institute and the National Insti-

tute for Nanotechnology (NINT). We would like to

thank the Canadian National High Field NMR Centre

(NANUC) for their assistance and use of the facilities.

The operation of NANUC is funded by the Canadian

Institutes of Health Research (CIHR), the Natural

Science and Engineering Research Council of Canada

(NSERC), and the University of Alberta.

References

1 Prusiner SB (1998) Prions. Proc Natl Acad Sci USA 95,

13363–13383.

2 Cosseddu GM, Agrimi U, Pinto J & Schudel AA (2007)

Advances in scrapie research. Rev Sci Techol 26, 657–

668.

3 Belay ED & Schonberger LB (2002) Variant Creutz-

feldt–Jakob disease and bovine spongiform encephalop-

athy. Clin Lab Med 22, 849–862.

4 Miller MW & Williams ES (2004) Chronic wasting dis-

ease of cervids. Curr Top Microbiol Immunol 284, 193–

214.

5 Collinge J, Whitfield J, McKintosh E, Beck J, Mead S,

Thomas DJ & Alpers M (2006) Kuru in the 21st cen-

tury – an acquired human prion disease with very long

incubation periods. Lancet 367, 2068–2074.

6 Almer G, Hainfellner JA, Brucke T, Jellinger K, Klein-

ert R, Bayer G, Windl O, Kretzschmar HA, Hill A,

Sidle K et al. (1999) Fatal familial insomnia: a new

Austrian family. Brain 122, 5–16.

7 Westergard L, Christensen HM & Harris DA (2007)

The cellular prion protein (PrP(C)): its physiological

function and role in disease. Biochim Biophys Acta

1772, 629–644.

8 Caughey B & Baron GS (2006) Prions and their part-

ners in crime. Nature 443, 803–810.

9 Watts JC & Westaway D (2007) The prion protein fam-

ily: diversity, rivalry, and dysfunction. Biochim Biophys

Acta 1772, 654–672.

10 Zomosa-Signoret V, Arnaud JD, Fontes P, Alvarez-

Martinez MT & Liautard JP (2008) Physiological role

of the cellular prion protein. Vet Res 39, 9.

11 Bremer J, Baumann F, Tiberi C, Wessig C, Fischer H,

Schwarz P, Steele AD, Toyka KV, Nave KA, Weis J

et al. (2010) Axonal prion protein is required for

peripheral myelin maintenance. Nat Neurosci 13,

310–318.

12 Pasupuleti M, Roupe M, Rydengard V, Surewicz K,

Surewicz WK, Chalupka A, Malmsten M, Sorensen OE

& Schmidtchen A (2009) Antimicrobial activity of

human prion protein is mediated by its N-terminal

region. PLoS ONE 4, 7358–7359.

13 Lee KS, Raymond LD, Schoen B, Raymond GL, Kett

L, Moore RA, Johnson LM, Kett L, Moore RA, John-

son LM et al. (2007) Hemin interactions and alterations

of the subcellular localization of prion protein. J Biol

Chem 282, 36525–36533.

14 Tagliavini F, Forloni G, Colombo L, Rossi G, Girola

L, Canciani B, Angeretti N, Giampaolo L, Peressini E,

Awan T et al. (2000) Tetracycline affects abnormal

properties of synthetic PrP peptides and PrPSc in vitro.

J Mol Biol 300, 1309–1322.

15 Vogtherr M, Grimme S, Elshorst B, Jacobs DM, Fiebig

K, Griesinger C & Zahn R (2003) Antimalarial drug

quinacrine binds to C-terminal helix of cellular prion

protein. J Med Chem 46, 3563–3564.

16 Hafner-Bratkovic I, Gaspersic J, Smid LM, Bresjanac

M & Jerala R (2008) Curcumin binds to the a-helicalintermediate and to the amyloid form of prion protein

– a new mechanism for the inhibition of PrPSc accumu-

lation. J Neurochem 104, 1553–1564.

17 Mok SWF, Thelen KM, Riemer C, Bamme T, Gultner

S, Lutjohann D & Baier M (2006) Simvastatin prolongs

survival times in prion infections of the central nervous

system. Biochem Biophys Res Commun 348, 697–

702.

18 Frid P, Anisimov SV & Popovic N (2007) Congo red

and protein aggregation in neurodegenerative diseases.

Brain Res Rev 53, 135–160.

19 Wishart DS, Knox C, Guo AC, Eisner R, Young N,

Gautam B, Hau DD, Psychogios N, Dong E, Bouatra

S et al. (2009) HMDB: a knowledge base for the human

metabolome. Nucleic Acids Res 37, D603–D610.

20 Lakowicz JR (2006) Principles of Fluorescence Spectros-

copy, 3rd edn. Springer, New York.

21 Mayer M & Meyer B (2001) Group epitope mapping by

saturation transfer difference NMR to identify segments

of a ligand in direct contact with a protein receptor.

J Am Chem Soc 123, 6108–6117.

22 Christen B, Hornemann S, Damberger FF & Wuthrich

K (2009) Prion protein NMR structure from Tammar

wallaby (Macropus euginii) shows that the B2–A2 loop

is modulated by long-range sequence effects. J Mol Biol

389, 833–845.

23 Yang M, Xi X & Yang P (2008) Thermodynamic analy-

sis of fluorescence enhancement and quenching theory

equations. Front Chem Chin 3, 254–261.

PrP binds thiamine R. Perez-Pineiro et al.

4012 FEBS Journal 278 (2011) 4002–4014 ª 2011 The Authors Journal compilation ª 2011 FEBS

24 Matsunaga Y, Peretz D, Williamson A, Burton D, Me-

hlhorn I, Groth D, Cohen FE, Prusiner SB & Baldwin

MA (2001) Cryptic epitopes in N-terminally truncated

prion protein are exposed in the full-length molecule:

dependence of conformation on pH. Proteins 44, 110–

118.

25 Jackson GS, Murray I, Hosszu LLP, Gibbs N, Waltho

JP, Clarke AR & Collinge J (2001) Location and prop-

erties of metal-binding sites on the human prion pro-

tein. Proc Natl Acad Sci USA 98, 8531–8535.

26 Walter ED, Chattopadhyay M & Millhauser GL (2006)

The affinity of copper binding to the prion protein

octarepeat domain: evidence for negative cooperativity.

Biochemistry 45, 13083–13092.

27 Nicoll AJ, Trevitt CR, Tattum MH, Risse E, Quar-

terman E, Ibarra AA, Wright C, Jackson GS, Sessions

RB, Farrow M et al. (2010) Pharmacological chaperone

for the structured domain of human prion protein. Proc

Natl Acad Sci USA 107, 17610–17615.

28 Langella E, Impronta R, Crescenci O & Barone V

(2006) Assesing the acid–base and conformational

properties of histidine residues in human prion protein

(125–228) by means of pKa calculations and molecular

dynamics simulations. Protein Struct Funct Bioinform

64, 167–177.

29 Lonsdale D (2006) A review of the biochemistry,

metabolism and clinical benefits of thiamin(e) and its

derivatives. eCAM 3, 49–59.

30 Gangolf M, Czerniecki J, Radermecker M, Detry O,

Nisolle M, Jouan C, Martin D, Chantraine F, Lakaye

B, Wins P et al. (2010) Thiamine status in humans and

content of phosphorylated thiamine derivatives in biop-

sies and cultured cells. PLoS ONE 5, e13616.

31 Thom JY, Davis RE & Icke GC (1986) Protein binding

of thiamine in human plasma. Int J Vitam Nutr Res 56,

189–195.

32 Parkhomenko IM, Strokina AA, Pilipchuk SI, Step-

anenko SP, Chekhovskaia LI & Donchenko GV (2010)

Existence of two different active sites on thiamine

binding protein in plasma membranes of synaptosomes.

Ukr Biokhim Zh 82, 34–41.

33 Ganapathy V, Smith SB & Prasad PD (2004) SLC19:

the folate ⁄ thiamine transporter family. Pflugers Arch

447, 641–646.

34 Tichopad A, Pfaffl MW & Didier A (2003) Tissue-spe-

cific expression pattern of bovine prion gene: quantifi-

cation using real-time RT-PCR. Mol Cell Probe 17,

5–10.

35 Moudjou M, Frobert Y, Grassi J & La Bonnardiere C

(2001) Cellular prion protein status in sheep: tissue-

specific biochemical signatures. J Gen Virol 82, 2017–

2024.

36 Bjorndahl TC, Zhou GP, Liu XH, Perez-Pineiro R,

Semenchenko V, Saleem F, Acharya S, Bujold A,

Sobsey C & Wishart DS (2011) Detailed biophysical

characterization of the acid-induced PrPC to PrPB con-

version process. Biochemistry 50, 1162–1173.

37 Delaglio F, Grzesiek S, Vuister GW, Zhu G, Pfeifer J

& Bax A (1995) NMRPipe: a multidimensional spectral

processing system based on UNIX pipes. J Biomol

NMR 6, 277–293.

38 Johnson BA (2004) Using NMRView to visualize and

analyze the NMR spectra of macromolecules. Methods

Mol Biol (NY) 278, 313–352.

39 Wishart DS, Bigam CG, Yao J, Abildgaard F, Dyson

HJ, Oldfield E, Markley JL & Sykes BD (1995) 1H, 13C

and 15N chemical shift referencing in biomolecular

NMR. J Biomol NMR 6, 135–140.

40 Marion D, Driscoll PC, Kay LE, Wingfield PT, Bax A,

Gronenborn AM & Clore GM (1989) Overcoming the

overlap problem in the assignment of proton NMR

spectra of larger proteins by use of three-dimensional

heteronuclear proton–nitrogen-15 Hartmann–Hahn–

multiple quantum coherence and nuclear Overhauser–

multiple quantum coherence spectroscopy: application

to interleukin 1.beta. Biochemistry 28, 6150–

6156.

41 James TL, Liu H, Ulyanov NB, Farr-Jones S, Zhang

H, Donne DG, Kaneko K, Groth D, Mehlhorn I, Prus-

iner SB et al. (1997) Solution structure of a 142-residue

recombinant prion protein corresponding to the infec-

tious fragment of the scrapie isoform. Proc Natl Acad

Sci USA 94, 10086–10091.

42 Goodsell DS, Morris GM & Olson AJ (1996) Auto-

mated docking of flexible ligands: applications of auto-

dock. J Mol Recognit 9, 1–5.

43 Guha R, Howard MT, Hutchison GR, Murray-Rust P,

Rzepa H, Steinbeck C, Wegner J & Willighagen EL

(2006) The blue obelisks interoperability in chemical

informatics. J Chem Inf Model 46, 991–998.

44 Morris GM, Huey R, Lindstrom W, Sanner MF, Belew

RK, Goodsell DS & Olson AJ (2009) AutoDock4 and

AutoDockTools4: automated docking with selective

receptor flexibility. J Comput Chem 30, 2785–

2791.

45 Schwieters CD, Kuszewski JJ, Tjandra N & Clore GM

(2003) The Xplor-NIH NMR molecular structure deter-

mination package. J Magn Res 160, 66–74.

Supporting information

The following supplementary material is available:

Figs. S1–S6. Stern–Volmer plots for data presented in

Table 1.

Fig. S7. 15N edited TOCSY-HSQC and NOESY-

HSQC strip plots for selected residues in thiamine

binding pocket.

Fig. S8. NMR spectra of thiamine (free) and thiamine

bound to shPrP.

R. Perez-Pineiro et al. PrP binds thiamine

FEBS Journal 278 (2011) 4002–4014 ª 2011 The Authors Journal compilation ª 2011 FEBS 4013

Table S1. List of human metabolites used in the

screening process.

This supplementary material can be found in the

online version of this article.

Please note: As a service to our authors and read-

ers, this journal provides supporting information

supplied by the authors. Such materials are peer-

reviewed and may be re-organized for online deliv-

ery, but are not copy-edited or typeset. Technical

support issues arising from supporting information

(other than missing files) should be addressed to the

authors.

PrP binds thiamine R. Perez-Pineiro et al.

4014 FEBS Journal 278 (2011) 4002–4014 ª 2011 The Authors Journal compilation ª 2011 FEBS