The Part of a Long Beta Hairpin from the Scrapie Form of the Human Prion

proteins STRUCTURE O FUNCTION O BIOINFORMATICS

The part of a long beta hairpin from the scrapie form of the human prion protein is reconstructed in the synthetic CC36 protein Vladislav Victorovich Khrustalev,1* Tatyana Aleksandrovna Khrustaleva,2 Kamil Szpotkowski,3 Victor Vitoldovich Poboinev,1 and Katsiaryna Yurieuna Kakhanouskaya1

1 Department of General Chemistry, Belarusian State Medical University, Dzerzinskogo, 83, Minsk, Belarus 2 Laboratory of Cellular Technologies, Institute of Physiology of the National Academy of Sciences of Belarus, Academicheskaya, 28, Minsk, Belarus 3 Department of Crystallography Center of Biocrystallographic Research, Institute of Bioorganic Chemistry, Polish Academy of Sciences, Z. Noskowskiego, 12/14, Poznan, Poland

ABSTRACT

Mechanisms of beta sheet formation by the human prion protein are not clear yet. In this work, we clarified the role of the region containing C-half of the second helix and N-half of the third helix of that protein in the process of alpha helix to beta sheet transition. Solid phase automatic synthesis of the original peptide (CC36: Cys179–Cys214) failed because of the beta hairpin formation in the region 206-MERVVEQMC-214 with a high beta strand potential. Using Met206Arg and Val210Arg substitutions, we increased the probability of alpha helix formation by that sequence. After that modification, the complete CC36 peptide with disulfide bond has been synthesized. Modified peptide has been studied by circular dichroism (CD) and fluorescence spectrography. According to the CD spectra analysis, the CC36 peptide contains 37% of residues in beta sheet and just 15% in helix. Thermal analysis under the control of CD shows that the secondary structure content of the peptide is stable from 58Cto808C. Dissociation of oligomers of the CC36 peptide finishes at 378C according to the fluorescence analysis. The CC36 peptide is able to bind Mn21 cations, which causes small temperature- associated structural shifts at concentrations of 2 – 10Á1026 M. Predicted beta hairpin of the CC36 peptide (two beta strands are: 184-IKQHTVT-190 and 197-TETDVKM-205) should be the part of a longer beta hairpin from the scrapie form of the prion protein (PrPSc). Analogs of the CC36 peptide may be considered as antigens for the future development of a vaccine against PrPSc.

Proteins 2016; 84:1462–1479. VC 2016 Wiley Periodicals, Inc.

Key words: prion protein; secondary structure; circular dichroism; tryptophan fluorescence; manganese; synthetic peptide.

INTRODUCTION a similarly infectious mechanism of the Alzheimer’s disease is being discussed.3 Interestingly, both pathological The major prion protein is a well-known protein that and normal prion proteins can form beta amyloid can undergo alpha helix to beta sheet transition and plaques together with Alzheimer’s peptide.4 form aggregates due to intermolecular beta sheet formation.1 Even though there are many other proteins that can undergo similar shifts and form aggregates involved Additional Supporting Information may be found in the online version of this in the development of chronic diseases (for example, article. Grant sponsor: BRFFR. pathological beta structural amyloid beta peptide is the The article is dedicated to the memory of Professor Eugene Victorovich Barkovsky cause of Alzheimer’s disease2), prion protein has a (18.05.1946–26.12.2015), who unfortunately did not see the result of the work that the authors planned together. unique feature: beta structural form of prion protein *Correspondence to: Vladislav Victorovich Khrustalev, Belarus, Minsk, 220029, (PrPSc) is able to cause a structural shift in the helical Communisticheskaya 7-24. E-mail: [email protected] form (PrPC) and promote aggregation.1 So, protease Received 13 February 2016; Revised 7 June 2016; Accepted 13 June 2016 Published online 18 June 2016 in Wiley Online Library (wileyonlinelibrary.com). resistant PrPSc is infectious.1 Currently, the possibility of DOI: 10.1002/prot.25090

1462 PROTEINS VC 2016 WILEY PERIODICALS, INC. Prion Peptide CC36 with Beta Hairpin

The exact molecular mechanism of PrPSc and its observed in conditions known to promote fibrillization: plaque formation is still unknown.5,6 Several regions of 200 mM NaCl and 4M urea in 5 mM sodium acetate at PrPC were shown to be able to form beta sheet in differ- pH 5.0, as well as after the reduction of the disulfide ent conditions. bond.1 However, the initial structure of that long peptide The structure of the N-terminal part of the prion pro- was the same as in the full-length prion protein. tein cannot be determined by X-ray and NMR meth- The synthetic peptide corresponding to the second ods.7,8 There are numerous imperfect tandem repeats in alpha helix of ovine prion protein consists mainly of ran- this part of the protein and they were shown to bind dif- dom coil at low pH.1 The synthetic peptide correspond- ferent metal cations.7,8 Peptides corresponding to those ing to the complete third helix of ovine prion protein at repeats are able to form beta sheet in the presence of pH 5 3.5 has a high content of random coil, while it metal cations in certain concentrations.9 should also have some amount of alpha helix, according The structured part of the prion protein is separated from to the CD spectra.1 tandem repeats by the highly conserved region with a run of In this study, we tested the structural state of the hydrophobic amino acid residues.10 The peptide correspond- human prion peptide entitled CC36 (Cys179–Cys214), ing to that region adopts an alpha helical conformation in which includes C-terminal half of the second helix, N- hydrophobic micelles.11 Theoretically, in water solutions, such terminal half of the third helix, and the loop between hydrophobic linear sequence of the full length protein may them, as well as disulfide bond between cysteine residues. form a core of intermolecular beta sheet. The peptide, which cannot form aggregates with an The only one region of prion protein with an additional intermolecular beta sheet (due to its shorter length and beta strand detected by crystallographic analysis is the pol- “attenuating” amino acid replacements) but keeps some ythreonine run (Thr191-Thr192-Thr193). That short addi- fragments of the beta hairpin from the infectious prion tional intermolecular beta strand has been found in dimers protein, may be considered as a candidate for vaccine of human prion protein possessing Met129 to Val129 ami- development. Antibodies against the pathological beta no acid replacement.12 structural Alzheimer’s peptide were shown to stimulate The structured part of the prion protein consists of the clearance of amyloid plaques in mice and patients three long alpha helices and two short beta strands. The with Alzheimer’s disease.17 So, antibodies against the length of the first beta strand may be extended under beta structural form of the human prion peptide may certain conditions. Peptides corresponding to this region also prevent the formation of aggregates by the patholog- are able to form beta sheet with each other.5,6 ical prion proteins. Antibodies against the fragment of The first helix is known to undergo a transition from beta hairpin from the pathological prion protein should alpha helix to random coil at low pH.14 This transition prevent the formation of the beta sheet between different from alpha helix to random coil is probably a necessary molecules of the PrPSc. Moreover, antibodies against the step for the next transition from alpha helix to beta beta hairpin of the PrPSc should decrease the rate of sheet.13 alpha helical prion proteins (PrPC) binding by the infec- According to the bioinformatic methods of secondary tious PrPSc and break the circle of the PrPC conversion structure prediction, the amino acid sequence of the sec- into the pathological infectious form. Immunization by ond alpha helix demonstrates a high potential of beta an antigen based on the CC36 peptide may be effective, strand formation.14 However, the peptides corresponding at least, in the initial period of the development of cer- to the second alpha helix are helical, and they are not form- tain transmissible prion diseases. ing beta sheet even in the presence of metal cations or at It was shown that manganese (II) cations bind prion pro- different pH.15 The second alpha helix is connected to the teins18,19 and stabilize the structure of both PrPC and third alpha helix by the disulfide bond. Short (6–7 amino PrPSc.20,21 Most of the studies on the interactions between acid residues in length) synthetic peptides from the second metal cations and prion protein fragments are concentrated and the third helices connected with each other by the on the N-terminal tandem repeats of the protein. Since man- disulfide bond were shown to exist in the form of beta ganese is one of the “modern” pollutants (the manganese- sheet.16 However, most of the short peptides from the containing compound—Methylcyclopentadienyl manganese prion protein may somehow form a beta sheet in certain tricarbonyl—is used as a gasoline additive), it is important to conditions,5,6 as well as other short hydrophobic peptides. check the possibility of its interactions with the fragment of According to one of the recent studies,1 the complete prion protein that is prone to structural shifts. Elevated man- domain composed of the second and the third helices is ganese levels have been reported in areas of high scrapie inci- the real carrier of the prion protein structural instability. dence.21 It was shown that prion protein demonstrates up to Indeed, the transition from alpha helix to beta sheet has 10-fold better survival in model soils contaminated with been reported for a long recombinant peptide composed manganese.21 Moreover, increased manganese content in the of full-length second and third helices (and the loop diet was shown to cause growth in the PrP expression level in between them) corresponding to the sequence of ovine the brain.21 Therefore, manganese can increase the transmis- prion protein.1 The structural shift in that domain was sibility of the pathological prion protein. The data from

PROTEINS 1463 Khrustalev et al. animal models provides evidence that prion disease leads to Both CD and fluorescence spectra have been measured an elevated manganese content in the blood and in some at different temperatures with the aim of detecting struc- areas of the brain.22 The same results have been obtained in tural shifts. CD spectra have been recorded in each the human population.23 Oneofthepossibleexplanations experiment at temperatures from 58Cto808C with a for this link between prion disease and manganese blood level step of 58C. Fluorescence spectra have been recorded in is that prion disease leads to the elevated expression of the each experiment at temperatures from 288Cto538C with divalent metal transporter 1 (DMT-1) protein that is respon- a step of 18C. Both CD and fluorescence spectra were sible for the manganese ions uptake.21 On one hand, envi- constant at each temperature. ronmental manganese may increase the success of infectious Van’t Hoff plots have been built in the standard coor- prion disease transmission; on the other hand, even subtle dinates: “ln(Keq)” has been put on the Y axis, while 26 prion disease may cause the elevation of manganese uptake in “1/T” has been put on the X-axis. Slopes of such the respiratory system and intestines leading to the increase of dependencies (which are equal to “-Ea/R”) have been its blood level.22 It is important to check whether manganese calculated by MS Excel. Then the values of the enthalpies ions in physiological and slightly elevated concentrations can (DH, kJ/mole) for each detected structural shift have beboundbytheCC36peptideandwhethertheyareableto been obtained. cause small structural shifts or even aggregation and intermo- Native gel electrophoresis has been performed at the lecular beta sheet formation. pH level equal to 4.4 (in the beta-alanine acetate buffer system) since the CC36 peptide has its isoelectric point (pI) in the slightly basic medium (near 8 according to the computational prediction with the protein isoelectric MATERIAL AND METHODS point calculator available via http://isoelectric.ovh.org/). The automatic solid-phase peptide synthesizer Together with the CC36 peptide, we used the horse myo- “Symphony” (Protein Technologies, Inc.) has been used for globin with pI 5 7.1 and the molecular weight of 17.8 the synthesis of both original and modified CC36 peptides. kDa, as well as cytochrome C with pI 5 10.2 and the Quality control has been performed with the help of HPLC molecular weight of 12.4 kDa (Serva Feinbiochemica (Agilent 1200) and MS (Shimadzu LCMS-2010) analyses. Dalton Standards kit 39064). We used agarose gel, stain- These procedures have been done commercially by the ing (amido black) and destaining solutions from the Peptide 2.0 Company (http://peptide20.com/). Cormay gel protein 100 kit (cat. no. 6-048). The running Circular dichroism (CD) spectra were collected on the J- time was equal to 15 min at the amperage of 11 mA. The original algorithm for the secondary structure pre- 815 CD spectrometer (JASCO) equipped with a Peltier- diction entitled “PentaFOLD.xlsx” (http://chemres.bsmu. thermostatted cell holder. For all spectral analyses, we used by) is based on two propensity scales. The first one is an solutions of the CC36 peptide in 0.01M phosphate buffer, amino acid propensity scale (see Supplementary Material, pH 5 5.3. The final concentrations of MnSO in cuvettes 4 list “Propensity scales”). To build that scale, we analyzed were equal to 1Á1026M,2Á1026M,5Á1026M,1Á1025M, and the amino acid content of alpha helices, beta strands, 2Á1025M. We also used a solution of the CC36 without and random coil regions of 542 proteins from bacteria Mn21 ions as a control. The final concentration of the pep- with different average genomic GC-content. We used the tide was equal to 3.6Á1025M in all experiments, except the 21 same set of proteins that has already been described in one with the highest Mn concentration (in the last case 27 25 our work on the 3/10 helices properties. There was no it was 3.1Á10 M). The path length of the cuvette was equal bias in that set associated with the design of the previous to 2 mm. Spectra have been recorded by three channels in study. The maximal similarity level in each pair of amino each experiment with a step of 0.5 nm from 185 to 320 nm. acid sequences of those proteins is 25%. Mutational GC- An analysis of CD spectra was performed with the help pressure leads to the increase of the usage of amino acid 24 of the CAPITO web server. We used the results of spectra residues encoded by GC-rich codons (Gly, Ala, Arg, and (and not the under curve area) matching (see Supporting Pro).28 Mutational AT-pressure results in the increase of Information, lists “Mn 0 – Mn 20”). The CAPITO web Phe, Tyr, Met, Ile, Asn, and Lys usages because those server matches original spectra with all the spectra from amino acids are encoded by GC-poor codons.28 Due to 24 the PCDDB database. the influence of negative selection, proteins encoded by Fluorescence spectra were recorded with the help of GC-poor genes are enriched by Ile, Asn, and Lys, while “Solar CM2230” fluorometer. The wavelength for the they lack Ala.29 The proteins encoded by GC-rich genes fluorescence excitation was equal to 270 nm.25 We are enriched by Ala, while lacking in Ile, Asn, and Lys.29 recorded emission spectra from 300 to 400 nm. The The proteins from bacteria with an average GC-content maximum intensity of fluorescence was detected at the have been used together with proteins from GC-poor wavelength of 350 nm in all of the experiments. That is and GC-rich proteomes in our data set. The algorithm why we used the intensity of fluorescence at the wave- considers an average probability to be included in a cer- length of 350 nm for Van’t Hoff plots building. tain secondary structure element for five amino acid

1464 PROTEINS Prion Peptide CC36 with Beta Hairpin residues from a linear sequence. For example, the probabil- regions of a protein wherein the probability of alpha helix ity for His187 from the KQHTV pentapeptide to be includ- to beta strand structural shift is high. Predictions made by ed in a beta strand is equal to the average probability for “PentaFOLD.xlsx” should be good for short peptides for Lys, Gln, His, Thr, and Val to be found in a beta strand. which the influence of other parts of the protein is mini- The second method from the “PentaFOLD.xlsx” is mal. Indeed, distant parts of a protein usually induce the based on the pentapeptide propensity scale.30,31 In that formation of alpha helices in fragments where beta strand method, amino acids are divided into two groups. might have been formed. Hydrophobic amino acids (O) are: Ala, Pro, Gly, Phe, For ab-initio 3D-modeling, we used the PEP-FOLD Tyr, Met, Ile, Val, Leu, Cys, and Trp. Hydrophilic amino server.33 The most probable model has been chosen acids (W) are: Arg, Asn, Lys, Gln, Glu, Asp, His, Ser, and from the whole set of generated models using the results Thr. Based on this information there are 25 5 32 possible of CD spectra analysis. combinations of O and W in pentapeptides. We calculat- For the modeling of the CC36 dimer and hexamer, we ed abundances of each pentapeptide in alpha helices, used the HEX 8.0.0 program.34 As an input, we used the beta strands, and random coil. Then we converted them most probable 3D model of the CC36 peptide created by into an original propensity scale (see Supplementary the PEP-FOLD server. From the set of generated dimer Material, list “Propensity scales”).30,31 The algorithm models, we have chosen the most energetically favorable “PentaFOLD.xlsx” has been created to explain the forces one. That model has tryptophan residues completely acces- stabilizing the secondary structure elements, rather than sible for water solution but quenched by side chains of glu- for prediction of actual conformation state. Indeed, the tamic acid residues, C-terminal carboxylic groups, and algorithm does not use any homology modeling. The disulfide bond (it is in agreement with the results of trypto- main idea of the algorithm is that cases where both phan fluorescence analysis). To visualize the structure of probability scales predict the same element of the sec- hexamer, we docked the monomer to the dimer and have ondary structure are not very frequent. Alpha helices are chosen the most probable trimer. After that, we docked usually formed in case there is a cluster of amino acids two trimers to each other. Hex 8.0.0 program was also used with a high probability of being included in that element for the docking of the model of the CC36 peptide to the of secondary structure, or in the case of hydrophilic and structure of the wild-type prion protein (1HJM). hydrophobic amino acids being arranged in the appro- We also used the BION server35 to find all the possi- priate manner. Thus, the “PentaFOLD.xlsx” algorithm ble binding sites for Mn21 ions on the model of the provides two possible patterns of secondary structure dis- CC36 dimer created by the HEX 8.0.0 program. tribution: “alpha helical” and “beta structural.” We tested both of them in a set of 50 proteins (See Supplementary Material, list “Training set”) from humans and other RESULTS mammals with the maximal similarity level in each pair Secondary structure predictions for the of amino acid sequences equal to 25%. wild-type human major prion protein The “alpha helical” pattern (when alpha helices predicted by at least one of the scales are in favor) shows the For the prediction of secondary structure elements, we following sensitivity and specificity: 64.86% and 40.42% used our original propensity scales.30,31 Those scales are for alpha helix; 36.81% and 46.06% for beta strand; based on the data obtained from the analysis of three- 57.00% and 74.13% for random coil. The “beta structural” dimensional structures of proteins with the maximal pattern shows opposite sensitivity and specificity regarding similarity of their amino acid sequences equal to 25%.27 alpha helices and beta strands: 32.37% and 44.04% for So, we can state that the method we used is a kind of alpha helix; 67.75% and 35.96% for beta strand; 57.18% purely ab-initio method and not homology-based one. and 74.10% for random coil. Parameters of the “alpha heli- In Figure 1(a), one can see that prion protein should cal” pattern of “PentaFOLD” are worse but comparable be mostly beta structural since the usage of amino acids (alpha helices and random coil regions are predicted signif- known as beta sheet formers is high throughout its icantly better than beta strands) with the performance of length. In contrast, in Figure 1(b), one can see that prion the “NPS: @Consensus” server32 in the same set of pro- protein should be mostly alpha helical since the combi- teins: 67.03% and 59.06% for alpha helix; 50.80% and nations of hydrophilic and hydrophobic amino acid resi- 56.41% for beta strand; 68.77% and 76.52% for random dues throughout its length are similar to those found in coil. The “beta structural” pattern of the “PentaFOLD” alpha helices. The main reason for the instability of the shows better sensitivity to beta strands than even the “NPS: secondary structure of the prion protein is represented in @Consensus” server that is based on the simultaneous Figure 1: amino acids prone to form beta strands are usage of the best ab-initio methods for secondary structure arranged in such combinations in which they can form prediction (DPM; DSC; GORI; GORIII; GORIV; HNN; only alpha helices. PHD; PREDATOR; SIMPA96; SOPM; SOPMA).32 So, Among three alpha helices of the human major prion using the “PentaFOLD” algorithm, one may find some protein, the first one looks like the most stable one.

PROTEINS 1465 Khrustalev et al.

amino acids as threonine (6 residues), valine (3 residues), and isoleucine (2 residues). The surprising result is that the method based on pentapeptide propensity scale predicted alpha helix from Phe175 to Val180. Yet another pentapeptide (His187-Thr188-Val189-Thr190-Thr191) from the body of the second helix is recognized as an alpha helical pentapeptide because it has a characteristic combination of hydrophilic (W) and hydrophobic (O) residues: WWOWW.30 However, in Figure 1(b), there is a peak of beta strand potential in the middle of the second helix (Asn181–Thr183). Interestingly, according to the results of circular dichroism and crystallographic experiments, peptides corresponding to the second helix of the prion protein exist in mostly alpha helical conformation.15 So, the combinations of amino acids in the second helix stabilize its secondary structure: the corresponding peptide cannot form beta strand on its own. According to our hypothesis, interactions with other parts of the prion protein, includ- ing the third alpha helix, should help the second alpha helix undergo the transition from alpha helix to beta strand. Just C-terminal part of the third alpha helix (Tyr218 – Tyr226) is predicted to be alpha helix by the method based on amino acid propensities [Fig. 1(a)]. N-terminal and middle parts of the third helix demonstrate high beta strand potential, even though it is not as high as in the second helix. According to the method, considering combinations of hydrophilic and hydrophobic residues, there are just two pentapeptides with high beta strand Figure 1 potential in the third helix: Asp202-Val203-Lys204- Results of the secondary structure prediction of the structured part of Met205-Met206 (WOWOO) and Gln212-Met213-Cys214- human prion protein using the PENTAfold.xlsx algorithm with the help Ile215-Thr216 (WOOOW).28 The real conformation of (a) amino acid propensity scale and (b) pentapeptide propensity scale. Secondary structure elements from 1hjm PDB file are shown. state of a peptide corresponding to the third helix of the [Color figure can be viewed in the online issue, which is available at ovine prion protein is mostly unstructured, while some wileyonlinelibrary.com.] structured fragments are noticeable according to the CD spectrum.1 It is probable that the same situation will be Indeed, according to the method based on amino acid observed for the full-length peptide corresponding to the propensities [Fig. 1(a)], there should be an alpha helix third helix of the human prion protein since there are starting from Asp147 and ending with His153. According just three amino acid substitutions between them to the pentapeptide propensity scale [Fig. 1(b)], which (Val203 to Ile203; Met205 to Ile205; Glu219 to Gln219). differentiates just hydrophobic and hydrophilic side The above mentioned amino acid substitutions are rela- chains of amino acids, there may be alpha helix from tively conserved, so we decided to check whether the N- His140 to Tyr145 and from Met154 to Val161. Moreover, terminal half of the third alpha helix will form a beta there are two “helical” pentapeptides with centers in hairpin with the C-terminal half of the second helix. Tyr149 and Tyr150. Since the borders of the first alpha helix are from Ser143 to Asn153 (it is followed by 3/10 Construction and synthesis of helix from Met154 to Tyr157) in the wild-type (PDB ID: the CC36 peptide 1hjm) human prion protein, one may suggest that a peptide corresponding to the first alpha helix of the human The initial sequence of the CC36 peptide had just one major prion protein will have mostly alpha helical struc- amino acid substitution relative to the fragment of the ture at physiological pH. wild-type major prion protein of Homo sapiens.We The second alpha helix of the prion protein is not pre- replaced Phe198 by Trp198 (Fig. 2). This modification has dicted by the method based on amino acid propensities. been made with the aim to introduce a fluorophore in the Instead of the second alpha helix, the method predicts a peptide. We also planned to create a disulfide bond long beta strand [Fig. 1(a)]. This is not surprising because between Cys1 and Cys36 of the peptide, as there is such that alpha helix contains such beta strand preferring36 bond between Cys179 and Cys214 of the prion protein.

1466 PROTEINS Prion Peptide CC36 with Beta Hairpin

As one can see in Figure 3(a,c), Met28 to Arg28 and Val32 to Arg32 replacements resulted in the growth of alpha helix potential in the C-terminus of the modified CC36 peptide, according to the amino acid propensity scale. In Figure 3(b,d), one can see that those two replacements resulted in the growth of the random coil potential according to the pentapeptide propensity scale. However, the pentapeptide Glu29-Arg30-Val31-Arg32- Glu33 (WWOWW) shows a strong alpha helix potential. We also introduced the Val2 to Pro2 replacement to increase the chances of successful disulfide bond formation Figure 2 between Cys1 and Cys36 (Fig. 2). Amino acid sequences of the wild-type human prion protein, original The modified sequence of the third alpha helix of the CC36 peptide, and modified CC36 peptide. Amino acid replacements prion protein did not cause problems during the synthesis are shown bolded and underlined. Amino acid residues that required a double coupling during the automatic solid phase peptide synthesis are (see Fig. 2). However, the problem with bad amino acid shown in italic font and highlighted. [Color figure can be viewed in the coupling appeared again when the chain already contained online issue, which is available at wileyonlinelibrary.com.] 26 residues. Each next residue required a double procedure of coupling. Finally, the mixture of different peptides was However, the first version of the CC36 peptide has not obtained. According to the results of mass-spectroscopy been synthesized because of the problems that occurred (see Supporting Information, list “Mass spectrum”), there during the process of automatic solid-phase peptide syn- was a peak corresponding to the molecular mass of the thesis. Starting from the twelfth amino acid residue (from peptide with disulfide bond (4264) in one of the fractions the C-terminus) the coupling of each next amino acid with obtained by HPLC. The procedure has been repeated with the growing peptide was poor. Even the repeated procedure the aim of obtaining 5 mg of the modified CC36 peptide. of coupling did not help. At the end of the synthesis pro- Coming back to Figure 2, we have to highlight that beta cess, we obtained numerous different species of peptides, hairpin was formed by Val11 and Thr12 residues (the first but there was no peptide of the required molecular mass beta strand) with some other regions already synthesized (both before and after the oxidation of –SH groups with into CC36 (the second beta strand). However, this beta the aim of creating a disulfide bond). According to our hairpin either did not completely prevent the coupling of hypothesis, growing peptides formed beta hairpins on the new amino acids or it was not formed by each of the column. Because of this, N-terminal NH groups were 2 numerous growing peptide chains. Some part of the grow- poorly accessible for activation and peptide bond forma- ing CC36 chains incorporated all of the amino acid resi- tion with supplied amino acids. As a result, different grow- dues in the correct order. Anyway, according to the results ing chains incorporated different amino acids. of the synthesis experiment we can suggest the existence of We failed to synthesize the first version of the CC36 beta hairpin in the final peptide. C-terminus of the modi- peptide. This experiment clearly showed us that the mid- fied peptide should not participate in the beta strand for- dle part of the third helix of the human prion protein is mation (unlike in the original sequence) while the region able to form beta structure. The beta structure is formed before Thr12 should make beta hairpin with the region sit- if there are no other parts of the prion protein interact- uated somewhere between Thr14 and Lys26. ing with that sequence. Our next step was to overcome the problem caused by beta hairpin formation with the Circular dichroism and fluorescence help of amino acid sequence modification. analysis of the modified CC36 peptide To prevent beta hairpin formation, we changed Val32 by Arg32 and Met28 by Arg28 (Fig. 2). Valine is the Circular dichroism spectra obtained with the help of strongest beta strand former.36 Methionine is known as Jasco J-815 spectrograph (Fig. 4) have been analyzed by alpha helix former34 while its beta strand potential, the CAPITO web server.22 Using that server, we con- according to our scale, is close to the alpha helix poten- verted original spectra in mdegs to spectra in mean resi- tial (0.3693 vs. 0.3745). We changed both Met28 and due elipticity. The closest spectra to the one for modified Val32 to arginine residues with the aim of creating a lin- CC36 peptide from the PCDDB belongs to pepsinogen. ear sequence “RERVR” in which positively charged side The percentage of amino acid residues in secondary chains of arginine residues would repulse each other. structure elements for pepsinogen is as follows: 15% in Indeed, in a beta strand side chains of amino acids are alpha helices, 37% in beta strands, and 48% in random always interacting in “i – i 1 2” manner.37 So, we tried coil regions. In the modified CC36 peptide, there should to avoid such interactions. Another benefit of arginine is be 5 amino acid residues in alpha helix, 13 residues in in its high alpha helix potential according to our scale beta strands, and 17 residues in random coil. Interesting- (0.4109). ly, because of mathematical rounding, the sum of three

PROTEINS 1467 Khrustalev et al.

Figure 3 Results of the secondary structure prediction of the original (a, b) and modified (c, d) CC36 peptide using the PENTAfold.xlsx algorithm with the help of amino acid propensity scale (a, c) and pentapeptide propensity scale (b, d). Secondary structure elements from 1hjm PDB file are shown. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.] above mentioned numbers is 35 instead of 36. In our that there are no shifts in secondary structure content opinion, there should be an even number of amino acid for the modified CC36 peptide between 58 and 808C (See residues in the beta hairpin. Probably, the real number of Supplementary Material, list “Mn 0”). amino acids in the beta sheet of the CC36 peptide is 14 The fluorescence of the single tryptophan residue is instead of 13. Thus, the structure of the modified peptide strongly quenched in the CC36 peptide. The quantum yield contains both alpha helix and beta hairpin. Based on of the tryptophan fluorescence is about 1.1%. However, the this, short alpha helix should occupy the position near spectrum can still be detected with the help of Solar the C-terminus of the peptide. The position of beta hair- CM2230 spectrofluorometer (Fig. 6). The peak of the spec- pin has been clarified with the help of ab-initio 3D trum is at 350 nm. This fact provides evidence that Trp20 modeling performed by the PEP-FOLD server.32 residue is situated in the hydrophilic environment (it is The model with the closest numbers of amino acid completely accessible to the solvent).25 Indeed, in the 3D residues in helix and beta strands is shown in Figure 5. model of the modified CC36 peptide, the Trp20 residue is In that model, cysteine residues from N- and C-termini completely accessible to the solvent, while carboxylic of the peptide are situated near each other, there is a sin- groups of Glu18 and Glu22 are situated near the Trp20 gle 3/10 helix made from 3 amino acid residues (Arg30- side-chain (Fig. 5). Indeed, carboxylic groups are known as Val31-Arg32) accompanied by the turn (Glu33-Gln34- quenchers of the tryptophan fluorescence. Met35), as well as two relatively long beta strands 7 ami- Thermal analysis of the modified CC36 peptide shows no acid residues in length each forming beta hairpin that there is a structural shift at 31–378C (Fig. 7). How- (Ile6 – Thr12 and Thr21 – Met27). Tryptophan residue ever, according to the CD spectra analysis, that shift is (Trp20) is not included in the beta strand, but it is situ- not associated with changes in secondary structure con- ated near its border (Fig. 5). Thermal analysis revealed tent. Using Van’t Hoff plots (see Supporting Information,

1468 PROTEINS Prion Peptide CC36 with Beta Hairpin

Figure 4 Circular dichroism spectra for the modified CC36 peptide at 258C in 0.01M phosphate buffer, pH 5 5.3. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.] list “Van’t Hoff plots”) we show that the enthalpy of disulfide bond is also known to quench tryptophan fluo- such structural shift is rather high (685 KJ/mol). Accord- rescence,25 so the orientation of peptides in the dimer is ing to our hypothesis, at lower temperatures the CC36 more likely to be “head-to-tail” (like that in Fig. 8) than peptide forms oligomers, while at higher temperatures “head-to-head.” As to the sixth model (DU 52470 those oligomers dissociate. In the oligomeric form of the KJ/mol), it keeps “head-to-tail” orientation, while loops peptide tryptophan residues should still be accessible to of the peptides are not connected with each other, and water molecules since the maximum of the fluorescence Trp20 residues are far away from possible quenchers spectra is at 350 6 2 nm at any temperature from 25 to from the other peptide chain. So, according to the 538C.23 (see Supporting Information, list “Native fluo- HEX 8.0.0 results, we can state that the most stable con- rescence spectra”). Since the intensity of fluorescence in formation of the CC36 dimer (Fig. 8) is in agreement monomeric form is higher than that in oligomeric form with the results of thermal analysis under the control of (Fig. 7), we can conclude that in oligomers, some amino tryptophan fluorescence measurement (Fig. 7). acid residues from one chain additionally quench the fluorescence of Trp20 from another chain. Indeed, such Native gel electrophoresis confirms the relationships can be found in the best model of CC36 existence of the CC36 peptide in the form dimer made by the HEX 8.0.0 server (Fig. 8). of an oligomer at a room temperature In the best HEX 8.0.0 model of the CC36 peptide dimer (DU 52529 KJ/mol), Trp20 is situated near the According to the results of the native gel electrophoresis, side-chain carboxylic group of Glu33 and C-terminal car- there is just one band corresponding to the CC36 peptide boxylic group of Cys36 (Fig. 8). The second best model (Fig. 9). So, at room temperature, the CC36 peptide forms a is less energetically favorable (DU 52498 KJ/mol) while certain oligomer and not a mixture of oligomers of different it keeps similar “head-to-tail” orientation with Glu33 orders. The molecular weight of the CC36 oligomer is from one peptide situated near Trp20 from another one. higher than 17.8 kDa. Indeed, the band corresponding to The third (DU 52486 KJ/mol) and the fourth the horse myoglobin that has a weight of 17.8 kDa migrated (DU 52483 KJ/mol) models are also built in “head-to- to the longer distance than the CC36 peptide. However, the tail” orientation while Trp20 in them is not quenched by difference in the distance between the CC36 and horse myo- amino acids from different peptide chain. Another orien- globin is almost the same as the difference in the distance tation of monomers (head-to-head) appears to be less between myoglobin and cytochrome C. So, if the molecular energetically favorable (DU varies from 2475 KJ/mol to weight of cytochrome C is 12.4 kDa, the molecular weight 2455 KJ/mol among models 5 and 7–10). Indeed, the of the CC36 peptide should be near 23.2 kDa.

PROTEINS 1469 Khrustalev et al.

The molecular weight of the CC36 monomer is 4.264 kDa, so the weight of its pentamer is 21.32 kDa, the weight of its hexamer is 25.584 kDa. It is more likely that we are dealing with hexamer since the distance between the bands for cytochrome C and myoglobin is longer than it would be if their pI values were identical (at pH 5 4.4 cytochrome C has higher positive charge than myoglobin). The possible structure of the CC36 hexamer is shown in Figure 10. Monomers are arranged in the “head- to-tail” manner in the quadruple core of that hexamer, and tryptophan residues are situated on the outer surface.

The influence of Mn21 ions on the structure of modified CC36 peptide We used five final concentrations of Mn21 ions in both circular dichroism and fluorescence analyses. In both cases (1Á1026M,2Á1026M,5Á1026M,1Á1025M, and 2Á1025M), the final concentrations have been established at the lowest temperature and then thermal analysis has been performed for the CC36 peptide solution. Results of CD spectra matching (with the CAPITO web server) showed that there were no shifts in the secondary structure content at the lowest (1Á1026M) and highest (2Á1025M) concentrations of Mn21 ions. At those concentrations of Mn21 cations, the secondary structure content of the CC36 peptide was the same as in the absence of Mn21. Moreover, fluorescence spectra analyses showed that there were no structural shifts associated with changes in Trp20 fluorescence. At the concentration of Mn21 ions equal to 2Á1026M, there was a shift from the initial state to the lower amount of residues in alpha helix (that is, from 15% to 9%, or 5 to 3 amino acids), and to the higher amount of amino acid residues in beta strands (that is, from 37% to 41%, or 14 to 16 amino acids). This small helix-to-sheet shift occurred at temperatures from 20 to 408Cifwe analyze the CD signal at 192 nm (Fig. 11). If we use the data from spectra matching by the CAPITO, then the shift occurred at 408C. According to the analysis of the Van’t Hoff plots (see Supporting Information, list “Van’t Hoff plots”), the enthalpy of this shift is equal to 188 kJ/ mol. The changed structure of the CC36 peptide is even closer to the 3D model represented in Figure 5 than the initial one. Indeed, the shortest length of an alpha helix is 4 amino acid residues, only 3/10 helix can demonstrate a

Figure 5 The model of CC36 peptide constructed by the PEP-FOLD algorithm, which contains the closest percentage of residues in beta sheet and helix to the results obtained by CD spectra analysis. 3/10 helix is shown in pink; beta strands are shown in yellow; turns are shown in blue; random coil fragments are shown in gray. The side chains of the tryptophan residue (Trp20) are also shown. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]

1470 PROTEINS Prion Peptide CC36 with Beta Hairpin

Figure 6 Spectra of fluorescence for the CC36 peptide in 0.01M phosphate buffer, pH 5.3, in the presence of 5Á1026M Mn21 at 288C, 358C, and 448C. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.] length of 3 amino acid residues. Fluorescence spectra anal- CC36 peptide did not dissociate at higher temperatures ysis showed that there were no structural shifts somehow because of the stabilizing influence of bound Mn21 ions. If affecting the intensity of Trp20 fluorescence from 25 to it is so, then secondary structure changes happened in the 538C. From this data, we can assume that oligomers of area that is relatively distant from Trp20 positions in the

Figure 7 The dependence between the temperature and the intensity of fluorescence at 350 nm of the CC36 peptide in 0.01M phosphate buffer, pH 5 5.3. Each marker corresponds to the intensity of fluorescence at a given temperature. Different markers are used to show fluorescence intensity before the shift (blue diamonds), during the shift (green triangles), and after the shift (red squares). [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]

PROTEINS 1471 Khrustalev et al.

was probably associated with the inclusion of Met27 into one beta strand and Ile6 into the opposite one. The explanation of the role of Mn21 binding in this structural shift can be as follows. At lower temperatures, Mn21 cations have been bound by individual atoms from side chains of amino acid residues while other ligands have been

Figure 8 The model of the CC36 peptide dimer created by the Hex 8.0.0 program. 3/10 helices are shown in pink; beta strands are shown in yellow; turns are shown in blue; random coil fragments are shown in gray. All the possible sites for Mn21 ions binding predicted by the BION algorithm are shown by gray dots. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.] Figure 9 Results of the native gel electrophoresis for the CC36 peptide together hexamer. So we can assume that initial alpha helix included with two markers of molecular weight (cytochrome C and horse myo- Arg28, Glu29, Arg30, Val31, and Arg32. Then Arg28 and globin). [Color figure can be viewed in the online issue, which is avail- Glu29 turned from alpha helix to random coil. This event able at wileyonlinelibrary.com.]

1472 PROTEINS Prion Peptide CC36 with Beta Hairpin

beta strands. However, after the structural shift shown in Figure 12, the percentage of amino acid residues in beta strands has become 41%. In Figure 13, one can see that the fluorescence intensity steeply decreases before the beginning of a shift. This decrease is associated with the increase in the length of beta strands. According to our hypothesis, Glu22 has not been included into the beta strand at lower temperatures, and it did not quench Trp20 fluorescence as strong as when it is included in the beta strand. Then the length of that beta strand has grown, and Trp20 fluorescence has been quenched. How- ever, the structural shift observed by CD analysis started the next structural shift observed by fluorescence analysis (the intensity of fluorescence grows from 35 to 448C). First, the secondary structure of the CC36 peptide had changed inside the oligomers by the way of the relatively small coil to sheet transition and only after the oligomers have dissociated. The enthalpy of the secondary structure change (182 kJ/mol) was lower than that for the mono- merization process (388 kJ/mol), according to an analysis of Van’t Hoff plots (See Supplementary Material, list “Van’t Hoff plots”). We need to highlight that in both monomeric and oligomeric states, the Trp20 residue has been completely accessible for water molecules since the maximal wavelength for its fluorescence was always at 350 nm (Fig. 6). At the concentration of Mn21 ions equal to 1Á1025M, the structural shift has been detected by CD spectra analysis at low temperatures (from 15 to 358C according to Figure 10 the analysis of CD signal at 191 nm, at 158C according The model of the CC36 peptide hexamer created by the Hex 8.0.0 to the spectra matching by the CAPITO). The nature of program. 3/10 helices are shown in pink; beta strands are shown in this shift shown in Figure 14 is the same as in the case yellow; turns are shown in blue; random coil fragments are shown in of 2Á1026M Mn21 concentration (helix to coil and coil gray. [Color figure can be viewed in the online issue, which is available to sheet), while the former one has been detected at at wileyonlinelibrary.com.] higher temperatures (from 20 to 408C). The enthalpy represented by water molecules (Fig. 8). At higher temper- according to the Van’t Hoff plots (see Supporting Infor- atures, some water molecules might have been lost, and mation, list “Van’t Hoff plots”) was equal to 146 kJ/mol. those positions might have been occupied by oxygen atoms In general, the temperature at which the structural shift from side chains situated nearby. Namely, the hydroxyl toward a higher amount of beta strands happens is decreasing with the increase of Mn21 concentration from group of Thr5 and amide group of Asn3 near the carboxyl 26 25 21 group of Glu29. At higher temperatures, these three groups 2Á10 M to 1Á10 M. Probably, at higher Mn concen- might move forward to each other and build the same trations, the same binding sites may be occupied by two coordination sphere for the single Mn21 ion. This move- ions, as has been seen in many X-ray structures. The 21 ment might cause the shift from alpha helix to 3/10 helix. presence of two Mn ions in the same binding site At the concentration of 5Á1026M of Mn21 ions, we increases the probability of the involvement of a higher observed first the shift in secondary structure content number of amino acid side chains in their coordination. (from 25 to 458C according to the analysis of CD signal On the other hand, there were no structural shifts at the 21 25 at 196 nm, at 308C according to the spectra matching by highest Mn concentration (2Á10 M). This phenome- the CAPITO) and then the sharp shift in fluorescence non should be linked with the complete saturation of all intensity of Trp20 (from 35 to 448C). First, we should the possible binding sites. highlight that the low-temperature CD spectra for CC36 26 21 in the presence of 5Á10 M of Mn is unique. Its sec- DISCUSSION ondary structure content is: 10% of alpha helix, 32% of beta strands and 58% of random coil. Such CC36 struc- The second and the third helices of the prion protein ture contains a smaller number of amino acid residues in have been suspected for their important role in the

PROTEINS 1473 Khrustalev et al.

Figure 11 The dependence between the temperature and the circular dichroism signal at 192 nm for the CC36 peptide in 0.01 M phosphate buffer, pH 5 5.3, in the presence of 2Á1026 MMn21 ions. Each marker corresponds to the intensity of fluorescence at a given temperature. Different markers are used to show fluorescence intensity before the shift (blue diamonds), during the shift (green triangles), and after the shift (red squares). [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]

Figure 12 The dependence between the temperature and the circular dichroism signal at 196 nm for the CC36 peptide in 0.01M phosphate buffer, pH 5 5.3, in the presence of 5Á1026M Mn21 ions. Each marker corresponds to the intensity of fluorescence at a given temperature. Different markers are used to show fluorescence intensity before the shift (blue diamonds), during the shift (green triangles), and after the shift (red squares). [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]

1474 PROTEINS Prion Peptide CC36 with Beta Hairpin

Figure 13 The dependence between the temperature and the fluorescence intensity at 350 nm for the CC36 peptide in 0.01M phosphate buffer, pH 5 5.3, in the presence of 5Á1026M Mn21 ions. Each marker corresponds to the intensity of fluorescence at a given temperature. Different markers are used to show fluorescence intensity before the shift (blue squares), during the shift (green triangles) and after the shift (red squares). To show several outlying points that cannot be used to build trends of linear dependence, we used small blue diamonds. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]

Figure 14 The dependence between the temperature and the circular dichroism signal at 191 nm for the CC36 peptide in 0.01 M phosphate buffer, pH 5 5.3, in the presence of 1Á1025M Mn21 ions. Each marker corresponds to the intensity of fluorescence at a given temperature. Different markers are used to show fluorescence intensity before the shift (blue diamonds), during the shift (green triangles), and after the shift (red squares). [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]

PROTEINS 1475 Khrustalev et al.

Figure 15 Four best conformations of the complex between the wild-type of the human prion protein (PDB ID: 1hjm) and the model of the CC36 peptide according to the HEX 8.0.0 docking results. The major island of beta structural potential is shown in blue; the minor island of beta structural potential is shown in green; the remaining part of the prion protein is shown in red; the remaining part of the CC36 peptide is shown in orange. structural shift from alpha helix to the beta sheet for a long The current study showed that there are two “islands” period of time.1,5,6,14,16 Short peptides from those heli- of beta structural potential in the third alpha helix of the ces have already been shown to be able to form beta prion protein. The strongest “beta structural island” is sheet.16 However, one cannot accept these results as direct situated in the middle of the third alpha helix (206- evidence of the hypothesis that these parts of the sequence MERVVEQMC-214). That sequence did not allow the are important for the structural shift in the full-length pro- original CC36 peptide to be synthesized by the way of tein. Most of the short relatively hydrophobic peptides solid phase automatic synthesis because of the beta hair- from prion protein and from other proteins as well are pins formed directly by the growing peptides. During the prone to form beta sheet.5,6 Another piece of evidence synthesis, the weaker “island” of beta structural potential came from the study on the whole domain composed of formed a hairpin with the previously described stronger both second and third helices.1 That domain is able to “island.” That weaker “island” should have a sequence of form beta sheet in certain conditions that are impossible in 197-TETDVKM-205. In the modified CC36 peptide, we living cells, but in relatively mild conditions it has an alpha decreased the beta strand potential of the stronger helical structure almost identical to the one in the full- “island” by two amino acid substitutions and the length prion protein.1 sequence “RERVR” (occurred in the place of “MERVV”)

1476 PROTEINS Prion Peptide CC36 with Beta Hairpin did not allow the formation of a beta hairpin. However, one can consider the analog of the CC36 peptide with the weaker “island” of beta strand potential has found the different pI level as a candidate for the development another partner to make a beta hairpin. That partner has of a vaccine against PrPSc. It was shown that antibodies been found somewhere in the second alpha helix. against the pathological (beta structural) Alzheimer’s According to our results, that partner should be the peptide are effective in slowing down the progression of sequence 184-IKQHTVT-190. That is how the modified this disease.39 So, the antibodies against the pathological CC36 peptide obtained its secondary structure. Even (beta structural) form of the prion protein should also though random coil occupies about one-half of the prevent the development of the disease. The problem length of that peptide, there is also a beta sheet com- with this strategy is the ability of pathological prion pro- posed of two antiparallel strands with seven amino acid teins to convert normal prion proteins into the scrapie residues in each and a very short alpha or 3/10 helix in form. So, one cannot use PrPSc as an antigen for vaccine the place of a modified sequence. development. The best way to overcome this problem is Interestingly, no problems with the synthesis of the to make a peptide that has a part of the beta structure of peptide corresponding to the full-length third helix of the full-length prion but cannot promote conversion of the ovine prion protein have been reported.1 It is likely normal prion proteins to PrPSc. According to the results that C-terminal part of the third helix possessing high of this study, the CC36 peptide really has a fragment of alpha helical potential influenced the structural state of the beta hairpin that is similar to the one from PrPSc. the middle part of that helix, which is the strong “island” There are no amino acid replacements in the beta strands of beta structural potential. Another explanation is in the of the model of the CC36 peptide relative to the wild- fact that there are amino acid substitutions in the major type human prion protein. There is just one amino acid beta structural island of the ovine prion protein (Val203 replacement in the loop between beta strands (Phe198 to to Ile203; Met205 to Ile205) relative to the wild-type Trp198) that will not be included in the future versions human homolog. Indeed, Ile has a little lower beta struc- of the vaccine peptide. Two amino acid replacements in tural potential than Val (0.541 vs. 0.565) while the beta the C-terminus of the CC36 peptide (Met206 to Arg206 structural potential of Met is lower (0.369) than that for and Val210 to Arg210) prevented the beta sheet forma- Ile. Probably, even these two conserved substitutions in tion during the synthesis of the peptide as well as the this region significantly changed the conditions in which aggregation of peptides during experiments. In our opin- the structural shift can happen. ion, these two substitutions destroyed the major island In the wild-type prion protein and in its domain com- of beta structural potential in the prion protein. The last posed of full-length second and third helices, one can amino acid replacement (Val180 to Pro180) helped to expect to find the formation of a longer beta hairpin make the disulfide bond in the peptide. So, three amino than that in the CC36 peptide. Indeed, both weak and acid replacements from the current study will be repeat- strong “islands” of beta structural potential from the ed in future peptides, while the central part of the anti- third helix should form a beta structure with the full- gen will be kept unmodified. length sequence of the second helix in the PrPSc type of The possible problem with the immunization by a the prion protein. beta structural prion peptide is as follows. The peptide Our results showed that there are multiple binding possessing the part of the core of PrPSc fibrils38 may sites for Mn21 cations on the CC36 peptide. At increased bind normal prion protein and stimulate its conversion concentrations of manganese, which are possible in case into the pathological form. Even though the peptide of intoxication, Mn21 ions are able to cause small struc- lacks the major island of beta structural potential, it may tural shifts of that peptide. Relatively small changes in interact directly with that island from the wild-type pri- the concentration of Mn21 ions lead to different types of on protein. According to the Hex 8.0.0 docking results, structural shifts. These shifts did not result in the aggre- there are just four conformations of the complex made gation of the CC36 peptide, but they may stimulate it in from the wild-type prion protein and the model of the the full-length prion protein that possesses unaltered CC36 peptide with the inner energy lower (DU 52591 major island of beta structural potential. Indeed, it is KJ/mol; 2537 KJ/mol; 2535 KJ/mol, and 2530 KJ/mol) likely that Mn21 or other cations can bind side chains of than that for the dimer of the CC36 peptide the same amino acid residues of the full-length PrPC (DU 52529 KJ/mol). In all these four conformations and PrPSc. So, researchers should be aware of the possi- (Fig. 15), the major island of the beta structural potential bility of the binding of metal cations not just by the N- of the wild-type prion protein does not interact with the terminal repeats, but also by the second and the third CC36 peptide. In the first three conformations [Fig. helices of the prion protein. 15(A–C)], the peptide mostly interacts with the second The CC36 peptide contains beta hairpin which should alpha helix of the prion protein while the beta strands of be part of the longer beta hairpin of the full-length the peptide are not parallel to the second helix of the PrPSc. According to our results, oligomers of the CC36 protein. In the fourth conformation [Fig. 15(D)], the peptide are completely dissociated at 378C. Theoretically, peptide is situated on the “back” side of the prion

PROTEINS 1477 Khrustalev et al. protein. Even though computer modeling did not give 11. Sauve S, Buijs D, Gingras G, Aubin Y. Interactions between the con- direct evidence of the possibility of the conversion of served hydrophobic region of the prion protein and dodecylphos- normal prion proteins by the CC36 peptide into the phocholine micelles. J Biol Chem 2012;287:1915–1922. 12. Lee S, Antony L, Hartmann R, Knaus KJ, Surewicz K, Surewicz pathological form, this possibility must be checked in WK, Yee VC. Conformational diversity in prion protein variants future experiments with modified vaccine peptides. influences intermolecular b-sheet formation. EMBO J 2010;29:251– Another strategy to obtain a stronger immune 262. response to that peptide is to make a synthetic construct 13. Honda RP, Yamaguchi K, Kuwata K. Acid-induced molten globule composed of the B-cell epitope of the peptide and T-cell state of a prion protein: crucial role of strand 1-helix 1-strand 2 segment. J Biol Chem 2014;289:30355–30363. epitopes of the widespread antigen, such as diphtheria 17,39 14. Groveman BR, Dolan MA, Taubner LM, Kraus A, Wickner RB, toxin. From this point of view, the CC36 peptide Caughey B. Parallel in-register intermolecular b-sheet architectures may be considered as a carrier of some B-cell and T-cell for prion-seeded prion protein (PrP) amyloids. J Biol Chem 2014; epitopes of PrPSc but not an infective prion peptide. 289:24129–24142. 15. Ronga L, Palladino P, Saviano G, Tancredi T, Benedetti E, Ragone ACKNOWLEDGMENT R, Rossi F. NMR structure and CD titration with metal cations of human prion a2-helix related peptides. Bioinorg Chem Appl 2007; Authors would like to acknowledge the help of Profes- 2007:10720 N. sor Wojciech Rypniewski, the Head of the Group of 16. Apostol MI, Perry K, Surewicz WK. Crystal structure of a human Structure-Function Relationship in Biological Molecules prion protein fragment reveals a motif for oligomer formation. J Am Chem Soc 2013;135:10202–10205. of the Institute of Bioorganic Chemistry of Polish Acade- 17. Yano A, Ito K, Miwa Y, Kanazawa Y, Chiba A, Iigo Y, Kashimoto Y, my of Sciences, who introduced them to each other and Kanda A, Murata S, Makino M. The peptide vaccine combined with gave a start to their international collaboration. prior immunization of a conventional diphtheria-tetanus toxoid vaccine induced amyloid b binding antibodies on cynomolgus mon- REFERENCES keys and guinea pigs. J Immunol Res 2015; 2015:786501. 18. Leach SP, Salman MD, Hamar D. Trace elements and prion diseases: a review of the interactions of copper, manganese and zinc with the 1. Adrover M, Pauwels K, Prigent S, de Chiara C, Xu Z, Chapuis C, prion protein. Anim Health Res Rev 2006;7:97–105. Pastore A, Rezaei H. Prion fibrillization is mediated by a native 19. Brazier MW, Davies P, Player E, Marken F, Viles JH, Brown DR. structural element that comprises helices H2 and H3. J Biol Chem Manganese binding to the prion protein. J Biol Chem 2008;283: 2010;285:21004–21012. 12831–12839. 2. Takashi RH, Tobiume M, Sato Y, Sata T, Gouras GK, Takashi H. 20. Choi CJ, Anantharam V, Martin DP, Nicholson EM, Richt JA, Accumulation of cellular prion protein within dystrophic neurites of amyloid plaques in the Alzheimer’s disease brain. Neuropathology Kanthasamy A, Kanthasamy AG. Manganese upregulates cellular pri- 2011;31:208–214. on protein and contributes to altered stabilization and proteolysis: 3. Roubaud BC, Varon C, Meqraud F, Salles N. Alzheimer’s disease: relevance to role of metals in pathogenesis of prion disease. Toxicol the infectious hypothesis. Geriatr Psychol Neuropsychiatr Vieil Sci 2010;115:535–546. 2015;13:418–424. 21. Davies P, Brown DR. Manganese enhances prion protein survival in 4. Morales R, Estrada LD, Diaz-Espinoza R, Morales-Scheihing D, Jara model soils and increases prion infectivity to cells. PLoS ONE 2009; MC, Castilla J, Soto C. 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J Mol Biol 1999;289:1163–1178. higher than the level of guanine in two-fold degenerated sites from

1478 PROTEINS Prion Peptide CC36 with Beta Hairpin

third codon positions of genes from Simplex- and Varicelloviruses 34. Ghoorah AW, Smail-Tabbone M, Devignes MD, Ritchie DW. Protein with G1C higher than 50%. J Theoretical Biol 2010;266:88–98. docking using case-based reasoning. Proteins 2013;81:2150–2158. 30. Khrustalev VV, Barkovsky EV. Stabilization of secondary structure 35. Petukh M, Kimmet T, Alexov E. BION web server: predicting non- elements by specific combinations of hydrophilic and hydrophobic specifically bound surface ions. Bioinformatics 2013; 15:805–806. amino acid residues is more important for proteins encoded by 36. Chou PY, Fasman GD. Prediction of the secondary structure of pro- GC-poor genes. Biochimie 2012;94:2706–2715. teins from their amino acid sequence. Adv Enzymol Relat Areas 31. Khrustalev VV, Khrustaleva TA, Barkovsky EV. Random coil struc- 1978;47:45–48. tures in bacterial proteins. Relationships of their amino acid com- 37. Bhattachargee N, Biswas P. Position-specific propensities of amino positions to flanking structures and corresponding genic base acids in the b-strand. BMC Sruct Biol 2010;10:29 N. compositions. Biochimie 2013;95:1745–1754. 38. Prigent S, Rezaei H. PrP assemblies: spotting the responsible regions 32. Combet C, Blanchet C, Geourjon C, Deleage G. NPS@: network in prion propagation. Prion 2011;5:69–75. protein sequence analysis. TIBS 2000;25:147–150. 39. Yano A, Miwa Y, Kanazawa Y, Ito K, Makino M, Imai S, Hanada N, 33. Shen Y, Maupetit J, Derreumaux P, Tuffery P. Improved PEP-FOLD Nisizawa T. A novel method for enhancement of peptide vaccination approach for peptide and miniprotein structure prediction. J Chem utilizing T-cell epitopes from conventional vaccines. Vaccine 2013; Theor Comput 2014;10:4745–4758. 31:1510–1515.

PROTEINS 1479