www.elsevier.com/locate/theochem

Journal of Molecular Structure: THEOCHEM 804 (2007) 9–15

Conformational studies on the prion protein 115–122 fragment

Viktoria Horvath a, Attila Kovacs b,*, Dora K. Menyhard c

a Structural Chemistry Research Group of the Hungarian Academy of Sciences at Eotvos University, H-1518 Budapest, Pf. 32, Hungaryb Hungarian Academy of Sciences, Budapest University of Technology and Economics,

Research Group for Materials Structure and Modeling, H-1111 Budapest, Szt. Gellert ter 4, Hungaryc Budapest University of Technology and Economics, Institute of General and Analytical Chemistry, H-1111 Budapest, Szt. Gellert ter 4, Hungary

Received 19 May 2006; received in revised form 18 September 2006; accepted 3 October 2006Available online 11 October 2006

Abstract

The conformational properties of the 115–122 region of the prion protein (PrP) were investigated by means of molecular mechanicsand DFT computations. From the results of the Monte Carlo Multiple Minimum conformational searches several representative groupscould be distinguished. Analysis of these and other characteristic structures like the a-helix and the geometry of the 115–122 region ofdissolved PrP (native) revealed the primary importance of N–H� � �O hydrogen bonding interactions in their stabilization, whereas the N–H� � �N interactions play only a marginal role. According to our PBE/TZVPP//RI-PBE/SVP DFT calculations, the isolated b-sheet anda-helical regular secondary structures and the native geometry are less stable than c-turn, which proved to be the global minimum geom-etry of the isolated 115–122 PrP fragment. The different solvation energies in aqueous solution, however, change the above picture. Theb-sheet structure can be considerable stabilized in water, becoming the most favoured at the PBE/TZVPP//RI-PBE/SVP level using theCOSMO solvation model. These results support a possible formation of the b-sheet structure of small PrP fragments involving the 115–122 region in aqueous solution, and in this way the proposed mechanism of their inhibition of the PrP(endogenous) fi PrP(pathogenic)conversion.� 2006 Elsevier B.V. All rights reserved.

Keywords: Prion; b-Sheet; Hydrogen bonding; DFT computations; Aqueous solution

1. Introduction

Transmissible neurodegenerative diseases (kuru andCreutzfeldt–Jakob disease in humans, scrapie and bovinespongiform encephalopathy in animals, etc.) are causedby a posttranslational misfolding of the responsible protein[1]. One of such proteins is the prion glycoprotein (PrP,Fig. 1), bound to the membranes of neuronal cells by a gly-cophosphatidylinositol anchor [2,3]. Two forms of PrPhave been observed which differ in their secondary struc-ture: the endogenous (PrPC) and the pathogenic one(PrPSc). Characteristics of PrPC are the high a-helix(43%) and minor b-sheet (3%) content, while in PrPSc thehelical content is decreased to 17–30% and the b-sheet con-

0166-1280/$ - see front matter � 2006 Elsevier B.V. All rights reserved.

doi:10.1016/j.theochem.2006.10.001

* Corresponding author. Tel.: +36 1 463 2278; fax: +36 1 463 3408.E-mail address: akovacs@mail.bme.hu (A. Kovacs).

tent is between 43% and 54%, depending on the length ofthe PrPSc fragment [4–6]. These different secondary struc-tures result in significantly different biochemical properties:PrPC is readily digested by proteinase K and soluble innon-denaturing solvents, while PrPSc is resistant to proteo-lytic digestion and highly insoluble. Moreover, the abnor-mal PrPSc isoform is assumed to catalyze the pathologicalPrPC fi PrPSc conversion [7] and has a tendency to aggre-gate forming amyloid plaques in the brain [8].

While the structure of PrPSc is elusive, that of PrPC hasbeen elucidated by NMR spectroscopy [9,10]. It containsan extended, largely unstructured N-terminal (23–90).Within this, residues 60–91 are able to bind Cu2+ in vivoselectively [11] with a consequence of becoming more struc-tured [12]. The 91–126 region is conformationally heteroge-neous, at low pH disordered. The globular domain 126–231consist of three helices (HA: 144–154, HB: 173–194, HC:

115-122

Fig. 1. The PrPC form of prion glycoprotein (115–122 region is marked).

10 V. Horvath et al. / Journal of Molecular Structure: THEOCHEM 804 (2007) 9–15

200–228) and a short two-stranded b-sheet (128–131, 161–164).

One of the main problems in prion research is thePrPC fi PrPSc conversion, in particular the region playingthe key role in it. Studies have been focused on differentregions of PrP. Model computational studies [13,14]implied first a possible role of the HA region (144–154),but recent experimental [15] and molecular dynamics(MD) [16] results indicated the stability of the HA helix.According to MD solution simulations [17], the helix struc-ture of HB (180–193) can break up in the middle resultingin two stable sub-helices. Other MD studies on thePrPC fi PrPSc conversion in the 109–219 region showed apromoting effect of low pH on the conversion [2,18].

The importance of the 90–127 region in thePrPC fi PrPSc conversion has been suggested in severalstudies [19–28]. Swietnicki et al., assumed considerableconformational changes in this region on the basis of CDspectroscopic results on the 90–231 domain of humanPrP [19]. Peretz et al., arrived to similar conclusions whencharacterizing the epitope regions of PrPC and PrPSc [20].The conformational polymorphism of the 106–126 frag-ment was investigated by CD spectroscopy by De-Gioiaet al. [22] and proved to be very sensitive on the environ-ment. They found that acidic pH and the presence of lipo-somes promote the formation of b-sheet. On the basis of1H NMR-restrained molecular mechanics studies Ragget al., suggested the importance of His111 (having amphi-philic ability and pH-dependent ionizable side-chain) forthe conformational mobility and heterogenity of the 106–126 region [21].

A probable important role of the 115–122 region in thePrPC fi PrPSc conversion is implied by a recent report ofChesebros and co-workers [24]. They found that small pep-tides containing the 115–122 fragment inhibit thePrPC fi PrPSc conversion (the catalysing effect of PrPSc is

vanished), although the mechanism is not yet cleared.The hypothesis is that these small peptides mimic the b-sheet structure of PrPSc and bind competitively to PrPSc

to block the conversion process. FT-IR measurements indi-cated indeed a high b-sheet content of the small peptides[24].

Our goal in the present study is to explore the conforma-tional properties of the 115–122 region (AAAAGAVV) bymolecular mechanics (MM) and quantum chemical calcu-lations. As most of the FT-IR measurements in Ref. [24]were carried out on suspensions due to the partial insolu-bility of the peptides, the secondary structures of the dis-solved molecules were not distinguished from those of thesolid. Therefore we paid special attention on the b-sheetconformer of 115–122, assessing its formation propensityon the basis of the computed relative energies in vacuumand in aqueous solution. In addition, comparison of ourresults on the 115–122 fragment with the geometry of thisregion in the 90–231 PrPC determined by NMR spectrosco-py [29] can serve to assess the energetic effects of the proteinbulk on the 115–122 region.

2. Computational details

The conformational search has been performed with theMonte Carlo Multiple Minimum (MCMM) method usingthe AMBER* force field [30,31] and the GB/SA solvationmodel [32] implemented in MacroModel [33]. We appliedMonte Carlo search with the random variation (withinthe range of 0–180�) of a randomly selected subset of alltorsional angles (a minimum of 2 and a maximum of 18torsions were altered). The perturbed structures were min-imized using the TNCG algorithm. The resulting minimumenergy structures were sorted by energy, and the uniquestructures within a 60 kJ/mol energy window above theglobal minimum were stored. The search jobs run upto

0

10

20

30

40

50

60

0 5 10 15 20 25 300

10

20

30

40

50

60

0 5 10 15 20 25 30

ΔE (kJ/mol)

ΔE (kJ/mol)

Terminal distance (Å)Terminal distance (Å)

a b

Fig. 2. Results of conformational searches performed in vacuum (a) and water (b), relative potential energy (kJ/mol) vs. terminal distance (A).

V. Horvath et al. / Journal of Molecular Structure: THEOCHEM 804 (2007) 9–15 11

100,000 steps. Selected characteristic structures have beenoptimized at the DFT level using the RI approximation[34] and the PBE functional [35,36] in conjunction with asplit valence plus polarization (SVP) basis set [37–39].The PBE density functional is known to provide goodaccuracy for a wide variety of systems including hydrogenbonds [40,41] and has been used in various studies of pep-tides, see e.g., Refs. [42,43].

In order to obtain more reliable energy data, single-point calculations at the PBE/TZVPP level (basis: triple-ze-ta valence plus two sets of polarization functions) havebeen performed on the RI-PBE/SVP optimized geometries.The large TZVPP basis set is advantageous for a reliableassessment of the energetics of weak (e.g., hydrogen bond-ing) interactions in biological systems. The solvent effectswere taken into account by single point calculations usingKlamt’s form of the conductor reaction field (COSMO)[44] at the PBE/TZVPP//RI-PBE/SVP level. All DFT cal-culations have been carried out using the Turbomole 5.6program [45].

1 The two selection criteria were the relative energy (possibly the lowest)and the number of hits (possibly the highest).

3. Results and discussion

We started our MCMM conformational search from thegeometry of the 115–122 region in the 90–231 PrPC frag-ment determined by NMR spectroscopy [29] (denoted inthe following as native). The chain was terminated bymethyl groups at both ends. For characterization of thenumerous local minima found we chose the distancebetween the nitrogen atom of the N-terminal residue andthe carbonyl oxygen atom of the C-terminal residue(parameter d) and the relative potential energy. The resultsof the conformational search are compiled in charts pre-sented in Fig. 2a and b.

The obtained global minimum conformation has a sim-ilar backbone to that of the native structure (with an rms of1.58 A excluding the terminal residues) even in the absenceof the conformational restrain of the rest of the proteinbulk, yet the energy difference is quite large (vide infra).

Additional MCMM searches starting from systematicallyconstructed a-helix, b-sheet and c-turn input structuresresulted in a very similar profile with the same global min-imum geometry (data not shown).

Analyzing the ca. 50,000 local minima on the basis ofparameter d, we distinguished ten main groups with consid-erable populations within which the structures had onlyminor differences (all atom rms of upto 0.6 A). Selectedexamples from each group1 (I–X) are depicted in Fig. 3.The relative energies and the characteristics of the intramo-lecular hydrogen bonding interactions of the selected repre-sentative structures are compiled in Table 1.

Seven of structures I–X contain a single turn around themiddle of the chain. The turn is stabilized by several hydro-gen bonding interactions connecting the two branches. Instructures V and X the C-terminal branch is bent fromthe nearly parallel position present in the other fivestructures.

Structures I, III, V, VII and VIII are c-turns at the res-idues Ala118, Gly119, Ala117, Ala120 and Ala120, respec-tively. The branches consist of mainly, often slightlydistorted, b-sheets. The b-sheet contents of the five struc-tures are 87.5%, 87.5%, 62.5%, 87.5% and 75%, respective-ly. We observed generally shorter (stronger) hydrogenbonds connecting the two branches than in the hydrogen-bonded five-membered rings of the b-sheet branches. Struc-tures IV and X are b-turns of type II at the residuesAla118–Gly119 and Ala117–Ala118, respectively. Thebranches are extended and generally disordered. Some b-sheet contribution (�25%) could be recognized only inthe N-terminal branch of structure X.

Two from the remaining three selected representativestructures (II, IX) are divided into three branches by dou-ble c-turns at residues Ala117, Ala120 and Ala116,Ala120, respectively. In structure II the branches are b-sheets (the two terminal ones slightly distorted) giving a

I. (γ-turn) II. III. IV. V. VI.

VII. (β-sheet) VIII. X.IX. α-helix native

Fig. 3. Selected characteristic structures from the MM conformational searches, the a-helical structure constructed for the 115–122 PrP fragment and thegeometry (denoted as native) found in the aqueous solution of 90–231 PrP.

12 V. Horvath et al. / Journal of Molecular Structure: THEOCHEM 804 (2007) 9–15

b-sheet contribution of �75% in this structure. In contrast,the middle branch in structure IX is disordered resulting ina reduced b-sheet content (�37.5%).

The last selected representative structure from theresults of the conformational search is the b-sheet (VI).Note that neither the a-helix nor the initial native conform-er were not present among the results of the conformation-al search implying their considerably higher energy for theisolated 115–122 fragment. We constructed the a-helixfrom the residues of the 115–122 PrP fragment and opti-mized its geometry together with that of the native con-former. While the DFT computations converged toreasonable a-helix and native geometries, the MM geome-try optimizations modified considerably the torsionalangles of both structures removing their original character.Therefore only the results of the DFT computations areincluded in Table 1.

Table 1 demonstrates the differences in the performanceof the three theoretical levels giving different stability orderfor most of the representative structures. The two DFT lev-els agree fairly in the computed relative energies. Notewor-thy deviations include the overestimated stability ofstructure II and the underestimated stability of structureVI by RI-PBE/SVP with respect to the PBE/TZVPP//RI-PBE/SVP level. In general, the MCMM derived datashow deviations similar in magnitude than those of theRI-PBE/SVP ones. For a few structures (II, V, VI, VII)the MM relative energies are even closer to the PBE/TZVPP//RI-PBE/SVP ones than those of the RI-PBE/

SVP calculations. Neither of the less sophisticated levelsagrees in the global minimum character of structure I:the RI-PBE/SVP level predicts it less stable by 10.4 kJ/mol, while the MM computations by 12.4 kJ/mol withrespect to the global minima at these latter levels. Note thatall the three theoretical levels disclose a superiority of theb-sheet structure (VI) of the 115–122 fragment in vacuumbeing higher by 21–62 kJ/mol than the respective globalminima. Moreover, the a-helix and native geometries seemto be considerably less stable explaining why they did notappear among the low-energy local minima of the conforma-tional search. The a-helix is higher in energy by 64 kJ/mol,while the native geometry by 103 kJ/mol. This considerablylow stability of the native structure (found in the solvated90–231 PrPC fragment [29]) seems to be too large to attributemerely to the absence of solute–solvent interactions andrefers to an important role of the protein bulk as well.

Inspecting the shape of the structures in Fig. 3, no cor-relation can be observed with the relative stabilities. E.g.,from the two most stable ones (at the PBE/TZVPP//RI-PBE/SVP level) structure I consists of two, whereas struc-ture II of three branches. More important for the relativestabilities of the conformers are the local interactions.From the three main local interactions, viz., steric,dipole–dipole and hydrogen bonding interactions, the lat-ter ones can be straightforwardly assessed by our computedgeometrical data.

The number and average length of the N–H� � �X(X = O, N) hydrogen bonds are compiled in Table 1. Most

Table 1Computed relative energies (DE, kJ/mol) and hydrogen bonding properties (number of hydrogen bonds and average hydrogen bond length rav, A) of the characteristic structures selected from the resultsof the conformational search

Structures

I II III IV V VI VII VIII IX X

c-turn b-sheet a-helix native

DEMM 0.0 �1.3 �4.5 �12.4 20.8 44.4 17.8 11.8 13.4 29.7DERI-PBE/SVP 0.0 �10.4 �4.0 4.8 22.0 51.5 30.4 33.8 28.2 50.8 65.8 105.9DEPBE/TVZPP

a 0.0 5.5 7.7 9.5 18.5 20.7 22.8 27.7 34.1 41.9 64.1 103.1DEsolv

MMb �102.1 �81.8 �100.4 �101.1 �109.5 �129.1 �116.4 �112.9 �122.1 �119.4

DEsolvPBE=TVZPP

c �72.9 �107.1 �71.6 �147.3 �143.2 �161.5 �139.2 �138.7 �131.7 �148.1 �175.8 �160.6

Hydrogen bonds:

N–H� � �O(ring)d 3 2 3 4 8 5 4 3 4 1rav 2.209 2.261 2.267 2.226 2.065 2.128 2.156 2.147 2.254 2.350N–H� � �O(loop)e 4 6 4 4 3 2 2 5 2 4 2rav 1.966 2.009 2.010 1.946 1.895 2.051 2.086 2.193 1.876 2.125 2.024N–H� � �N 1 1 7 5rav 2.611 2.483 2.368 2.377

a From single-point calculations on the RI-PBE/SVP optimized geometries.b Solvation energy in water from MM geometry optimizations using the GB/SA solvation model.c Solvation energy in water from single-point calculations on the RI-PBE/SVP optimized geometries using the COSMO model.d Number of hydrogen bonds between neighbouring (geminal) C=O and N–H groups forming hydrogen-bonded five-rings.e Number of hydrogen bonds between non-neighbouring C=O and N–H groups forming loops.

V.

Ho

rvath

eta

l./

Jo

urn

al

of

Mo

lecula

rS

tructu

re:T

HE

OC

HE

M8

04

(2

00

7)

9–

15

13

14 V. Horvath et al. / Journal of Molecular Structure: THEOCHEM 804 (2007) 9–15

hydrogen bonds, found on the basis of the van der Waalscutoff criteria, are of the N–H� � �O=C type. We distin-guished between N–H� � �O interactions between neighbour-ing N–H and C=O groups forming five-membered rings,and interactions between non-neighbouring groups form-ing loops. The former hydrogen bonds are generally weak-er because of the larger H� � �O distances (cf. Table 1). Thehydrogen bonds between non-neighbouring N–H andC=O groups are shorter by 0.1–0.3 A, thus they result inlarger stabilization. Analyzing the data of Table 1 we canfind a fair correlation between the number and strengthof hydrogen bonds and the PBE/TZVPP//RI-PBE/SVPstability order. E.g., structures I–IV have four–six loop-type hydrogen bonds, whereas up to three only of theweaker ring-type ones. The opposite situation can beobserved for the less stable structures V–VIII and X. Theb-sheet contains eight relatively short hydrogen bonds infive-membered rings. In IX and in the a-helix the loop-typehydrogen bonds are relatively weak as shown by their larg-er average length. The least stability of the a-helix andnative conformers is in agreement with the found smallnumber and weak character of their N–H� � �O interactions.

N–H� � �N interactions obeying the van der Waals cutoffcriteria were found only in the four least stable structures.According to the H� � �N distances they are very weak,hence their contribution to the overall stability of the con-formers must be marginal.

The effect of aqueous medium on the relative stabilitiesof the secondary structures of 115–122 PrP was assessed bytwo approaches: by the GB/SA solvation model [32] in theMCMM calculations re-optimizing the geometries and bysingle point calculations using Klamt’s form of the conduc-tor reaction field (COSMO) [44] at the PBE/TZVPP//RI-PBE/SVP level.

The GB/SA calculations resulted in a distribution of theminimum structures in water similar to that in vacuum (cf.the two charts in Fig. 2). The global minimum conformer(structure IV) is the same in the two phases with an allatom rms of 0.10 A. On the other hand, the energy differ-ences between the global and local minima are considerablysmaller in the aqueous solution than in vacuum. E.g., the b-sheet type structures (terminal distance �25 A) appear inwater by 23 kJ/mol higher than the global minimum,whereas in vacuum this energy difference is 55 kJ/mol.

The solvation energies collected in Table 1 are generallysomewhat above 100 kJ/mol. We note the somewhat largerstabilization of the b-sheet in water compared the otherstructures. We observed also a decrease in the populationof the groups of type II, V, VIII, IX and X structures, prob-ably as a result of considerable changes in the geometryupon solvation effects.

The solvation energies obtained using the COSMOmodel show a somewhat different picture. At this level,the solvation energies of the b-sheet, a-helix and nativestructures are between �160 and �176 kJ/mol, much largerthan those of most other structures. As a consequence, theb-sheet turns to be the most preferred conformer in the

aqueous solution at the PBE/TZVPP//RI-PBE/SVP level.These results support the propensity of small peptides con-taining the 115–122 fragment to form b-sheet in aqueoussolution which is the prerequisite of their assumed bindingto this region of the PrPSc protein and inhibition of thePrPC fi PrPSc conversion [24].

The considerable stabilization of the native structure inthe aqueous solution gives a more reliable information onthe effect of the protein branch, proving a much less effectthan the energy difference obtained for the isolatedstructures.

4. Conclusions

The focus of the present study was on the conformation-al properties of the 115–122 fragment of the prion protein(PrP) in vacuum and in aqueous solution. Our work wasinitiated by a recent hypothesis of Chesebro et al., whofound that small synthetic peptides containing the 115–122 region of PrP can inhibit the PrPC fi PrPSc conversion[24]. Our results show that the b-sheet conformation of the115–122 fragment can be stable in aqueous solution fulfill-ing the prerequisite of its binding to the same region ofPrPSc and blocking the PrPC fi PrPSc conversion.

The high stability of the b-sheet in aqueous solution isdue to its larger solvation energy compared with the struc-tures being preferred in vacuum. The latter structures arestabilized by a large number of strong N–H� � �O hydrogenbonding interactions between non-neighbouring residues,while in the b-sheet only weak interactions between theneighbouring groups can be formed. This deficiency isovercompensated by the gain in the solvation energy inwater.

Acknowledgements

This research was initiated by Prof. Istvan Hargittai andwe thank him for advice and discussion. Financial supportfrom the Hungarian Scientific Research Foundation(OTKA No. T046183, T042933) and computational timefrom the National Information Infrastructure Develop-ment Program of Hungary is gratefully acknowledged.A.K. thanks the Bolyai Foundation for support.

Appendix A. Supplementary data

Cartesian coordinates of the discussed structures opti-mized at the RI-PBE/SVP level. Supplementary data asso-ciated with this article can be found, in the online version,at doi:10.1016/j.theochem.2006.10.001.

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