A role for His155 in binding of human prion peptide144–167 to immobilised prion protein

Biochemical and Biophysical Research Communications 362 (2007) 695–699 www.elsevier.com/locate/ybbrc

A role for His155 in binding of human prion peptide144–167 to immobilised prion protein J. Richard Hesp *, Neil D.H. Raven, J. Mark Sutton Health Protection Agency, Porton Down, Salisbury, Wiltshire SP4 0JG, UK Received 31 July 2007 Available online 17 August 2007

Abstract The interactions and conformational changes that lead to the conversion of the normal prion protein (PrPc) to its pathogenic form, PrPsc, are still being elucidated. Using Surface Plasma Resonance (SPR), we provide evidence that a synthetic peptide (PrP144–167) corresponding to residues comprising the a helix 1–b strand 2 domain of PrPc is able to interact and bind to immobilised recombinant human PrP (rHuPrP) in a dose-dependent manner. The interaction is pH dependent with an increase in binding observed as the pH is lowered, particularly between pH 6.5 and pH 5.5 suggesting a specific role for His155 in the interaction, confirmed by covalent modification of this residue in the peptide with diethylpyrocarbonate (DEPC). Circular dichroism analysis of PrP144–167 revealed no secondary structure motifs across the pH range investigated. Possible pH related structural changes of immobilised rHuPrP are also discussed with regard to the increased affinity for PrP144–167.  2007 Elsevier Inc. All rights reserved. Keywords: Prion; Transmissible spongiform encephalopathy; Creutzfeldt-Jakob disease; Surface plasmon resonance; Peptides

Transmissible spongiform encephalopthies (TSEs) are a group of invariably fatal neurodegenerative diseases and include scrapie in sheep, bovine spongiform encephalopathy (BSE) in cattle and Creutzfeldt-Jakob disease (CJD) in humans. A new variant form of the human disease (vCJD) emerged in the 1990s, which has been linked to the consumption of BSE-infected meat products [1]. Given the possibility that a significant percentage of the UK population has been exposed to infected meat the potential exists of much higher levels of vCJD occurring in the population. The transmissible agent of vCJD and all TSE disorders is proposed to be proteinaceous in nature and contain no nucleic acid component [1]. In the protein-only hypothesis put forward by Prusiner [2] the key step in the pathogenesis of the disease is the conversion of the prion molecule from a conformation which is primarily a helical (PrPc) to one of b-sheet (PrPsc). NMR on PrPc in solution confirmed its a-helical conformation for both human and *

Corresponding author. Fax: +44 (0) 1980 611310. E-mail address: [email protected] (J.R. Hesp).

0006-291X/$ - see front matter  2007 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2007.08.050

bovine species [3–5], and more recently for a number of other mammalian species [6]. In all the above species, PrPc consists of a flexible, extended N-terminal domain (residues 23–120) including the highly conserved octarepeat, PHGGGWGQ. This repeat sequence has been shown to have a high affinity for copper ions [7] thus indicating a possible role for the prion protein in the regulation of copper in the cellular environment [8]. The C-terminal domain (residues 125–228) is more structured with three a helices, a short antiparallel b-sheet and a single disulphide bridge between two of the helices. The crystal structures for ovine [9] and human PrP [10] have been determined with the latter solved as a dimer which revealed detailed information on the secondary structures involved at the interface. However, it is not known how significant this is, in the conversion of PrPc to PrPsc. In contrast to PrPc, PrPsc is characterised by its insolubility in physiological conditions, where it can exist as multimers from a few repeating units to much larger fibril type structures, with the highest relative levels of infectivity found in the nonfibrillar particles [11]. The insolubility of PrPsc has

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prevented its crystal structure from being solved, but a combination of electron microscopy and predictive modelling is starting to reveal additional structural information about this form [12]. The aim was to use SPR technology to identify short peptides which were capable of interacting with PrPc. Here we have identified one such peptide, residues 144–167, which spans helix, a1, and b strand, b2, in PrPc; a region which undergoes substantial conformation changes during the conversion into the pathogenic form [20,21]. The identification of such peptides, and the possible modes of binding involved, could lead to new insights into the conversion process and therefore to new molecules which could inhibit the process in vivo. Materials and methods Synthetic peptides. Synthetic peptides were synthesised using standard solid phase chemistry and were desalted prior to use (Activotec Ltd.). Peptides were based on a consensus of mammalian sequences [13] and are numbered in relation to human PrP (HuPrP). Residues different to HuPrP are underlined. PrP(C)126–146 (C-GGYMLGSAMSRPIIHFGNDYE), PrP(C)144–167 (C-DYEDRYYRENMHRYPNQVYYRPVD), PrP(C)144–167(C) (C-DYEDRYYRENMHRYPNQVYYRPVD-C), PrP144–167 DYEDRYY RENMHRYPNQVYYRPVD), PrP179–202 (CVNITIKQHTVTTTTKGE NFTETD and PrP 214–231(CITQYQRESQAYYQRGAS). PrP(C)126–146, PrP(C)144–167 and PrP(C)144–167(C) cysteine variants were synthesised to allow alternate methods of immobilisation if required. Peptides, PrP144–167 and variants thereof, and PrP179–202, were dissolved in SPR running buffer (10 mM MOPS, pH 7.4, with 150 mM NaCl and 0.005% P20 surfactant (Biacore) unless otherwise stated. Peptides, PrP(C)126–146 and PrP 214–231, were not soluble at neutral pH and therefore were first dissolved in a small volume of alkali as a concentrated stock, the pH slowly adjusted to neutral with acetic acid and then diluted with SPR running buffer to the working concentration. Aliquots of each peptide solutions were stored at 20 C until use. Surface plasma resonance (SPR) analysis. A Biacore X instrument was used to analyse protein: peptide interactions by means of SPR [14]. The ligand, recombinant human PrP (rHuPrP) (Abcam Ltd.) was covalently immobilised onto a CM5 chip (carboxymethylated dextran surface) via primary amine groups [15] using the manufacturers protocol. Immobilisation of rHuPrP was carried out at a protein concentration of 35 lg/ml, diluted in 20 mM phosphate, pH 7.0, at a flow rate of 5 ll/min to achieve a surface density of approximately 9000 RU for the initial screening experiments (Fig. 2). For subsequent experiments the surface density was reduced to between 3000 and 4000 RU. Unless otherwise stated, the reference flow cell on the chip consisted of a control ligand, adenylate kinase, purified as described in Vonrhein et al. (1998) [16] immobilised at 65 lg/ml diluted in 10 mM sodium acetate, pH 4.5. SPR running buffer was 10 mM MOPS, pH 7.4, with 150 mM NaCl and 0.005% P20 surfactant (Biacore) unless otherwise stated. The peptide solutions (analyte) were injected over the surface at 25 C at a flow rate of 5 ll/ml in running buffer (association phase). For the dissociation phase peptide solutions were replaced with running buffer. The chip surface was regenerated using 50 mM Glycine, pH 9.5, injected at 5 ll/ml for 30 s. Each time course of the SPR analysis (sensorgram) was blanked against the response detected in the reference flow cell. BIAevaluation 3.0 SPR kinetic analysis software (Biacore) was used to analyse the binding reactions. Immobilisation of PrP(C)144–167 to a CM5 chip was carried out using the peptide’s N-terminal cysteine in conjunction with the reagent, 2-(2pyridinyldithio)ethaneamine (PDEA) as described in the manufacturer’s protocol (Biacore). Diethylpyrocarbonate (DEPC) modification of peptides. N-carbethoxylation of histidine residues was carried out using DEPC. Peptides were dissolved in 10 mM MES, pH 6.5 + 150 mM NaCl to a final concentration of 1.2 mg/ml. DEPC was prepared as a 100 mM stock solution dissolved

in water on the day of use and added at a 10-fold molar excess over peptide. After 30 min at room temperature the reaction was quenched by the addition of a molar excess of histidine and buffer exchange on a NAP 10 column (Pharmacia) pre-equilibrated in the above buffer. To confirm that modification had occurred the absorbance at 240 nm of modified and unmodified peptide was measured and the percentage of modified peptide calculated using an extinction coefficient of 3200 M/l for N-carbethoxyhistidine [17].

Results Peptide analytes Peptides were chosen from several regions of the prion molecule associated with secondary structure elements identified within the C-terminal domain [11]. The relative positions of the peptides within HuPrP that were chosen for analysis are shown in Fig. 1. Peptides PrP(C)126–146 and PrP(C)144–167 overlap and span the region from b strand, b1, through to and inclusive of b strand, b2. Peptide PrP179–202 includes the helical motif, a2 and PrP 214–231 consists of the C-terminal end of a3 through to the GPI anchor addition point [18]. Analysis of the binding of prion-derived peptides to rHuPrP using surface plasma resonance (SPR) Comparison of the binding of the peptides to immobilised rHuPrP on the sensor surface is shown in Fig. 2, using a high immobilisation density of ligand to establish the binding specificity of the peptides. Peptides PrP(C)126–146, PrP179–202, and PrP 214–231 showed minimal binding to the sensor surface. In contrast binding events were observed with peptide PrP(C)144–167, with the association and dissociation phases of the interaction demonstrating a high initial on/off rate, respectively. Bulk shift effects were observed for the peptide solutions and these were compensated for by the use of a reference surface containing an ‘inert’ protein, recombinant adenylate kinase [16], immobilised at a similar density to rHuPrP. A variant of peptide PrP(C)144–167, containing an additional C-terminal cysteine (peptide N181

N197

SS HuPrP 23

51

β1 α1 β2 α2

91 Octarepeat region

126

α3

231

146 144

167 179

202 214

231

Fig. 1. Schematic representation of human PrP showing the relative positions of the PrP-derived synthetic peptides. PrP23–231, corresponding to the mature protein after post-translational processing, is represented by the contiguous white box. Open boxes indicate the positions of the b strands, hatched boxes indicate the a-helical regions with the position of the di-sulphide bond indicated above the contiguous white box. N-linked glycosylation sites are indicated by arrows.

J.R. Hesp et al. / Biochemical and Biophysical Research Communications 362 (2007) 695–699 PrP peptide

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(c)126 -146 (c)144-167 (c)144-167(c) 179-202

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Fig. 2. Peptide PrP(C)144–167 shows binding to immobilised rHuPrP using SPR analysis. Sensorgrams of peptide binding to rHuPrP; 200 lM of each peptide was injected at 5 ll/ml for 300 s. Of the peptides analysed only PrP(C)144–167 showed significant binding to immobilised rHuPrP under the conditions tested.

PrP(C)144–167(C)), was also assessed but showed reduced binding activity compared to PrP(C)144–167. Characterisation of the binding of peptide PrP(C)144–167 to rHuPrP To investigate the properties of peptide PrP(C)144–167 further, the analysis was repeated with a series of different concentrations of the peptide analyte applied to the sensor (Fig. 3A). A dose–response relationship between concentration of analyte and RU values was observed on the sensorgram. At the highest concentration of peptide (200 lM) the RU values showed a slight decrease towards the end of the inject phase (200–300 s) which is most likely attributable to the peptide approaching its limit of solubility. In an attempt to further define the association and dissociation phases, a Langmuir binding model was applied to the data to describe a 1:1 binding stoichiometry between analyte and ligand (with and without mass transfer effects). Analysis of the plot of residuals indicated that the models were a poor fit for the data, particularly at the start of the association and dissociation phases (data not shown). The application of a model which accounts for the presence of a heterogeneous ligand, which can occur due to the method of attachment of ligand to the chip surface, thus leading to parallel binding events occurring, resulted in an improved fit with a v2 value of 12.9 compared to a v2 of 43.8 for the 1:1 fit. However, analysis of the residual plot again indicated that although the residuals were relatively low they were not randomly distributed and therefore this model was also incorrect. Thus the apparent better fit of the parallel model was probably the result of a more complex model being applied [19]. To therefore determine if independent or non-independent binding reactions were occurring, analysis of the dissociation phase was performed

B

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Injection time 300s 30s

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Fig. 3. (A) The binding of PrP(C)144–167 to immobilised rHuPrP shows a dose–response relationship within the micromolar range. SPR was performed with a range of PrP(C)144–167 concentrations injected at 5 ll/ ml for 300 s. (B) Examination of the type of binding between PrP144–167 and rHuPrP indicates non-independent binding events occurring on the sensor chip. PrP144–167 peptide (200 lM) was injected over immobilised rHuPrP at 5 ll/ml for 30 and 300 s. The dissimilar dissociation curves indicate non-independent binding events thus confirming that models based on a simple 1:1 interaction between analyte and ligand are not applicable.

using long and short injection times (Fig. 3B). The dissociation rate was much slower after the long injection time indicating that binding events were not independent and that either bi-analyte or conformational change events were occurring on the chip surface [19]. Although the association/dissociation kinetic constants could not be determined due to the poor fit of models to the existing data sets, it can be observed that the affinity of peptide PrP(C)144–167 for PrP is within the high micromolar range at pH 7.4 in the presence of 150 mM NaCl as indicated by the high peptide concentrations required for binding. An alternate chip was prepared using peptide PrP(C)144–167 as the ligand, immobilised via its N-terminal cysteine to a CM5 chip using PDEA, and with PrP as analyte. However, rHuPrP showed a very strong tendency to bind to both modified and unmodified surfaces, possibly due to the molecule’s basic nature (predicted pI of 9.39; Compute pI/Mw; http://www.expasy.org/sprot/), and therefore this model was not investigated further. The effect of pH on the interaction between PrP144–167 and rHuPrP was investigated (Fig. 4A). Reducing the pH

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Relative response (RU)

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1400 1200 1000

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to selectively modify His155 to generate its N-carbethoxy derivative. Covalent modification had a profound effect on the binding capability of peptide PrP144–167 to rHuPrP; a population of peptide with 50% of histidine residues modified under the conditions tested (based on spectrophotometric measurements) resulted in a reduction in peak RU values from around 120 to 70 (Fig. 4B) demonstrating the importance of His155 in the binding. No decrease in absorbance at 275–280 nm was observed during the time course of the reaction indicating that the extent of Tyrosine modification by DEPC was minimal.

Time (s)

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Fig. 4. Effect of pH (A) and DEPC treatment (B) on the binding of PrP144–167 to rHuPrP. SPR was performed as described in Materials and methods with an ethanolamine blocked reference cell. (A) The peptides were prepared at a final concentration of 200 lM in their respective buffers for each pH examined. (B) DEPC derivitisation was performed using a 10fold molar excess of DEPC over peptide as described in Materials and methods.

between pH 8.0 and pH 7.0 resulted in a marginal increase in binding, however, lowering the pH between pH 6.5 and pH 5.5 resulted in approximately a twofold increase in peak RU values. Within the peptide the only residue where the ionisation state could change in the above pH range is His155 (pKa = 6.5). The pKa of other ionisable side chains within the peptide fall outside this pH range and therefore a change in the ionisation state of His155 would have a significant effect on the overall charge of the peptide. Attempts to assess lower pH values were unsuccessful due to the relative insolubility of the peptide under these conditions. Analysis of peptide PrP144–167 by far-UV circular dichroism (CD) spectroscopy revealed no secondary structure motifs (results not shown) suggesting that this peptide fragment of PrPc is likely to exist in solution as a random coil. Effect of DEPC modification of His155 on binding of peptide PrP144–167 to rHuPrP In order to further investigate the role of His155 in the binding of peptide PrP144–167 to rHuPrP, DEPC was used

Using the published structural information and consensus regions for mammalian PrPc the peptides chosen in this study were designed to span several key structural elements within the C-terminal domain (Fig. 1). Real time binding studies using surface plasmon resonance revealed that only peptide PrP144–167 interacted with immobilised rHuPrP. The importance of the peptide PrP144–167 region, comprising helix 1 and b strand 2, has emerged from a variety of published studies that suggest a significant role in the conversion of PrPc to PrPSc (e.g., [10,18]). Furthermore, It has been postulated that this region is the most probable site for conformational conversion to the b-rich form [20,21]. The binding studies described here indicate a specific and dose-dependent interaction of peptide PrP144–167 with rHuPrP. Although the precise mode of binding could not be determined the suggestion that the mode of binding may be non-independent is intriguing as it has been shown that the peptide PrP144–167 region lies at the interface of a PrPc dimer [10]. It is therefore possible that this peptide is interacting with the same region of the bound PrP leading to the formation of an ‘on-chip’ dimer with a comparable interface and a stabilised conformation. The substantial increase in binding of peptide PrP144–167 as a result of lowering the pH provides further information on the basis of the interaction. There was a negligible effect on binding within the pH range of pH 7.0 to pH 8.0, however, between pH 6.5 and pH 5.5 peak RU values doubled. Of the ionisable groups present within the peptide only the pK for the single histidine falls within this pH range, suggesting that this residue may be critical. No secondary structure for peptide PrP144–167 within the above pH range was apparent using CD analysis and therefore based on this observation, changes in affinity cannot be attributed to conformational changes of the peptide itself. Similar peptides from this region of murine PrP/ovine PrP [22] and human PrP [23] have also been analysed by CD which indicated that some regular structure was present, however, these peptides were extended at the N-terminus compared to PrP144–167. Further analysis of PrP144–167 using NMR would confirm whether any secondary structure was present within the above pH range. Although the increase in binding of PrP144–167 could not be attributed to changes in the structure of the peptide itself, another possibility

J.R. Hesp et al. / Biochemical and Biophysical Research Communications 362 (2007) 695–699

for the increased affinity of the peptide for PrP could be due to the acidic pH altering the conformation of the immobilised PrP to favour binding. A number of studies (e.g., [24–26]) have shown that an acidic pH environment results in changes in the secondary structure of PrPc supporting this possibility. Specifically the region around His155 was destabilised in acidic conditions and could represent the initial steps in the conversion to the b-sheet rich species [18]. Within peptide PrP144–167, the direct involvement of His155 in the binding to PrPc is supported by the demonstration that its covalent modification with DEPC results in a significant reduction in the binding of the peptide to immobilised rHuPrP. In the well conserved peptide PrP144–167 His155 is actually one of the least conserved amino acids with the residue present in human and bovine PrP but replaced with tyrosine in sheep, cervids and mouse (and with asparagine in hamsters). Mutations at this position in the hamster gene, substituting Tyr155 for Asn155, significantly reduced the formation of protease-resistant hamster PrP [27] thus suggesting a unique role of this position in the biology of the disease. The identification of a peptide ligand derived from a domain present at the human PrPc dimer interface or possible PrPc:PrPsc interface(s), that can bind to the non-infectious form could provide a possible lead for therapeutic intervention. Future work will focus on further defining the nature of the interaction between peptide PrP144–167 and PrPc and to expand the analysis to include its pathogenic variant, PrPsc.

[7]

[8] [9]

[10]

[11]

[12]

[13]

[14]

[15]

[16] [17] [18]

Acknowledgments [19]

Funding for the study was from the UK Department of Health. The views expressed in the publication are those of the authors and not necessarily those of the Department of Health. The authors gratefully acknowledge Prof. Ravi Acharya and Dr. Laurie Irons, University of Bath, for their expert advice and assistance with the CD analysis.

[20]

[21] [22]

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