Proc. Nail. Acad. Sci. USAVol. 84, pp. 4470-4474, July 1987Biophysics

Theoretically determined three-dimensional structure for therepeating tetrapeptide unit of the circumsporozoite coat proteinof the malaria parasite Plasmodium falciparum

(antigen/energy minimization/hydrogen bonding/molecular dynamics/protein conformation)

BERNARD R. BROOKS*, RICHARD W. PASTORt, AND FREDERICK W. CARSONt§*Division of Computer Research and Technology, National Institutes of Health, Bethesda, MD 20892; tBiophysics Laboratory, Office of Biologics Researchand Review, Food and Drug Administration, 8800 Rockville Pike, Bethesda, MD 20892; tDepartment of Chemistry, American University,Washington, DC 20016; and §Malaria Branch, Naval Medical Research Institute, Bethesda, MD 20814-5055

Communicated by Martin Karplus, February 2, 1987 (received for review September 18, 1986)

ABSTRACT A model for the three-dimensional structureof the repeating Asn-Pro-Asn-Ala tetrapeptide of the im-munodominant circumsporozoite protein of Plasmodiumfalciparum has been developed. A trial structure in the form ofa type I fi turn with asparagine side chains hydrogen-bondedto the backbone peptide linkages was used as a starting point.A repeating oligomer of this trial structure was modeled usingenergy minimization and molecular dynamics computer sim-ulations in conjunction with image boundary conditions. Themost stable structure generated is a right-handed 1238 helix,which is unlike any previously identified protein secondarystructure. The helix has 12 residues per turn, corresponding toan angle of twist of 1200 per tetrapeptide unit, and a pitchof 4.95 A, corresponding to a rise of 1.65 A per tetrapeptideunit. It is highly stabilized by extensive hydrogen bonding, witheach tetrapeptide unit acting as an acceptor for five hydrogenbonds and as a donor for five hydrogen bonds to residues inadjacent turns as well as having four weak internal hydrogenbonds. A number of nearly isoenergetic variations on the moststable structure that still retained the basic 1238 helical motifwere also discovered. The implications of these structures forvaccine development are discussed.

The recent determination of the sequence of the repeatingtetrapeptide of the circumsporozoite (CS) coat protein of thehuman malaria parasite Plasmodium falciparum (Pf) (1, 2) isof great medical, biochemical, and theoretical interest. Ma-laria, a parasitic disease spread by infected Anopheles mos-quitoes, has afflicted mankind throughout recorded history.More than half the world's population is exposed to malaria,especially in tropical areas (3). It is estimated that as many as300 million people have the disease worldwide and that itcontributes to the deaths of one million infants per year inAfrica alone (4, 5). The World Health Organization began acampaign to eradicate malaria in the 1950s. However, thedevelopment of resistance to insecticides by mosquitoes, theappearance of malarial strains resistant to prophylacticdrugs, political instability, and economic difficulties have ledto a resurgence of malaria in the last decade (6).The inability of modem medicine to control malaria, let

alone develop a cure suitable for widespread use, has ledmany investigators to conclude that the main hope for thefuture will lie in the development ofvaccines (5, 7, 8). Surfaceantigens of sporozoite and merozoite stages of Plasmodiacausing malaria have been considered as vaccine candidatesbecause of their exposure to the mammalian host's immunesystem prior to invasion of target cells. Particular attentionhas been paid to CS proteins, which are immunodominant

surface antigens ofunknown function produced in abundanceduring the sporozoite stage of the life cycle of these proto-zoan parasites (9, 10).The immunological properties of these proteins (10-15)

have stimulated molecular biological research aimed at vac-cine development. Thus, genes coding for CS proteins froma number of plasmodial species have been cloned andsequenced (reviewed in ref. 16). In every case, a portion ofthe protein contains a tandemly repeated peptide, whichprovides the immunodominant epitopes. Among these, therepeating tetrapeptide sequence of the Pf CS protein is thesimplest. In one strain the major reiterated unit is Asn-Ala-Asn-Pro repeated in tandem 37 times with 4 interspersedAsn-Val-Asp-Pro variants (1). Chemically synthesized andcloned peptide fragments of the repeating region have beenthe focus of intensive research as potential vaccine compo-nents, since Pf is the most dangerous of the human malarias(7, 8, 17-19).The unprecedented simplicity of the repeat of the Pf CS

protein encouraged the prospect of calculating its structure.This paper reports the results of a theoretical investigation ofthat structure using energy minimization and moleculardynamics methods to develop a three-dimensional computermodel.T

THEORY AND METHODSAn exhaustive search for the most stable conformation of aprotein, presumably the native one, is not feasible (20).Therefore, a variety of other computational search methodswere considered. The most reliable method for structureprediction of proteins currently available involves compari-sons with homologs for which x-ray crystallographic struc-ture determinations have been made (ref. 21 and referencescited therein). A search of the National Biomedical ResearchFoundation Protein Sequence Database (Release 9.0) re-vealed that 27 other registered proteins contain variations ofthe Pf repeated tetrapeptide. However, none of these havehad their three-dimensional structures determined, so thisapproach was not possible. Another method is to build theprotein systematically, residue by residue (22), or fromblocks of di- and tripeptides (23). However, current limits oncomputer memory and speed restrict such approaches to arelatively coarse grid sampling size or to a small number ofamino acids.

Assumptions. The approach used here is based on twopremises. First, every repeating tetrapeptide is assumed to

Abbreviations: CS, circumsporozoite; Pf, Plasmodium falciparum;6, angle of twist per tetrapeptide unit in degrees; d, rise or axialdisplacement per tetrapeptide unit in A.$This work was presented in part at the Sixth International Congressof Parasitology, Brisbane, Australia, August 25, 1986.

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The publication costs of this article were defrayed in part by page chargepayment. This article must therefore be hereby marked "advertisement"in accordance with 18 U.S.C. §1734 solely to indicate this fact.

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have the same conformation within an arbitrary helical form.This is reasonable if the environment of each tetrapeptideunit is similar to that of its neighbors or if the structure issufficiently stable that inhomogeneities in the environmentwill not disrupt it. This is the case, for example, in the mainchain of a-helices of native globular proteins. Second, it isassumed that the correct structure is accessible from a trialpeptide conformation by a low-energy pathway. This pro-vides a method for conformational searching using dynamicssimulations and energy minimizations within particular sym-metry constraint parameters. Taken together, these twoassumptions allow a systematic evaluation of the conforma-tional parameters in a straightforward manner. Operational-ly, the approach consists of three steps, which are discussedin turn.Development of a Trial Structure. To apply molecular

dynamics, reasonable starting conformations must be used.Conformational preference parameter probability calcula-tions [P, conformational parameter (subscripts: a, a-helix; (3,3 sheet; t, turn)] using a three-state model (24) indicated thatneither the a-helix nor (3-sheet conformations were likely forAsn-Pro-Asn-Ala (Pave = 0.83 and Pp,ave = 0.79), whereasa (-turn structure was probable (Ptave = 1.33). Informationcontent calculations (25) suggested that random coil and(3-turn conformations were probable.Based on these results, molecular models of Asn-Pro-Asn-

Ala in a number of (3-turn conformations were examined. Therange of possibilities was reduced by noting that asparagineoften appears at position 1 of (3 turns, whereas proline has agreat tendency to be in position 2 of type I and III ( turns (26).It was found that additional stability could be conferred onthe type I structure through hydrogen-bonding of the side-chain carbonyl group of each asparagine residue to thebackbone N-H group of the next asparagine residue in theC-terminal direction. Hence, that conformation was chosenas the trial structure.Method of Periodic Images. The method of images (27) was

developed to take advantage of any inherent symmetry of asystem. Generality is achieved by the explicit treatment ofonly one set of symmetrically unique primary atoms, in thiscase the atoms of a single tetrapeptide. Any interactionbetween these atoms and symmetrically related atoms iscalculated by the use of temporary image atoms, which aredefined by symmetric transformations in terms of transla-tions and rotations of the primary atoms. The positions of theimage atoms are recomputed from those of the primary atomsfor every energy evaluation and in this way symmetry isimposed as a rigid constraint.For periodic helical structures, the relationship between

repeating subunits may be specified by a simple set ofsymmetry transformations involving two free parameters: 9,the angle of twist per subunit, and d, the rise or axialdisplacement per subunit. For example, in an ideal a-helix,neighboring residues are related by 9= 1000 and d = 1.45 A.Only d values of one sign need to be considered becausechanging the signs of 0 and d result in an equivalent helix. Inthe case of the repeated tetrapeptide, the upper limit of d isconstrained by the connectivity to be about 10 A. The twistcan take on any value, but there are combinations of 6 and dthat lead to unfavorable nonbonded contacts (for example, ifthe ratio of d to 6 is <0.01 A/degree). The value of 9 aloneis not sufficient to determine whether the helix will beleft-handed or right-handed nor will it determine how tightlywound it is about the helical axis for a given pitch. This comesabout because a number of physically different structures aredefined by the same mathematical transformation. For ex-ample, a left-handed starting structure with a twist of -160°could in principle undergo a transition to a right-handedstructure with a twist of +2000 and in an extreme case go toa structure with a twist of +5600. Analogous transitions were

observed in the course of this study. For this application,there was often only one physically reasonable twist for aparticular set of helical parameters, and at most there weretwo.Use of Molecular Dynamics. Each molecular dynamics

simulation based on the method of images was carried out inthe following manner from a particular starting structure: aninitial energy minimization of 100 steps was followed by 1 psof equilibration at 300 K (1000 steps of molecular dynamics)and then 500 steps of reminimization. All calculations wereperformed with CHARMM (Chemistry at HARvard Macro-molecular Mechanics) (27) with the parameter set PARAM19(28) in vacuo. A distance-dependent dielectric (e = r) with ashift potential terminating at 7.5 A was employed. Each setof calculational steps took =1.5 hr on an Apollo DN-600.

RESULTS

An extended ribbon-like helix with 9= 1800 and d = 5.0 A wasconstructed from the trial structure flanked by images oneither side within a io-A cutoff. Optimization caused only aslight change in conformation and yielded a structure with anenergy of -141.2 kcal/mol (1 kcal = 4.148 kJ) of tetramer,which had converged to within 0.01 kcal/mol. The notation9180d5.0 is used to refer to this structure. Other optimizedstructures will be named analogously.The tetrapeptide conformation in 9180d5.0 was then used

as a template for the generation of the helical structures9180d4.5 and 0180d5.5. Keeping 9 constant, a row of 9 = 1800structures with d ranging from 1.5 to 7.0 A in 0.5-A incre-ments was constructed by successive use of this stratagem.Employing the same approach, for a given value ofd the angle9 was then varied in increments of ±200 over the range 200 to3400, and final increments to 100 and 3500 were taken. In thisway the template was continuously deformed and =520right-handed and left-handed helices were constructed overessentially the entire range of d and 9. Distinct energy boundswere found, with structures at the extremes of d and 9suffering from bad van der Waals contacts (in low 0 and lowd structures) or insufficient stabilization (at high d). Coordi-nates and energies of these helices are available uponrequest.Two distinct minimum energy regions were found on this

grid and these were explored in increments of 50 for 9 and0.05 A for d. One of the minima was a right-handed,ribbon-like extended structure, 0170d2.8, with an energy pertetramer of -153.7 kcal/mol. The best helical structurecalculated was a right-handed, 12-residue-per-turn helix,0120dl.65, with an energy of -164.4 kcal/mol of tetramer.Deformations of d by ±0.05 A from the best structureresulted in an energy rise of 1.6 kcal/mol oftetramer whereasvariation of 9 by ±2.0° from the quadratically fitted minimum(118.90) resulted in an energy rise of 1.4 kcal/mol of tetramer.Table 1 contains the Cartesian coordinates and torsion

angles for the heavy atoms of 0120dl.65. The structure is anunprecedented 1238 helix with a pitch of4.95 A and a diameterof =17 A. The hydrogen bond network is shown in Fig. 1. Theside-chain amide groups of the asparagine residues areparallel to the helical axis, allowing for the formation ofinterrepeat H-bonds. Good hydrogen bonds to all othercarbonyl groups are also present. Each unit lines up with theones above and below it much as would be seen in a parallel(3 sheet, but with one section turned so that the threebackbone H bonds all point in the same direction, rather thanalternating. It may be seen from Table 1 that two of the 4 and4' torsion angle pairs (Asn-1 and Pro-2) are similar to those ofa parallel (3 sheet, whereas the other two (Asn-3 and Ala-4)are close to those of a left-handed a-helix, even though thestructure is globally right-handed. Fig. 2 shows a view downthe central axis of the helix. The faces of each turn form an

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Table 1. Cartesian coordinates (A) and dihedral angles(degrees) for a single Asn-Pro-Asn-Ala unit of the mostfavorable calculated structure for the repeat region ofthe Pf CS protein.

Residue Atom

Asn-1 NCACBCGOD1ND2C0

Pro-2 NCDCACBCGC0

Asn-3 NCACBCGOD1ND2C0

Ala-4 NCACBC0

(Asn-5 N

x

-3.079-3.320-2.096-2.375-2.307-2.702-4.498-4.345-5.724-6.203-6.832-8.020-7.651-6.568-6.614-6.251-6.085-7.306-7.179-7.136-7.104-4.817-4.507-4.077-2.898-3.254-1.728-1.554-0.907

y

-2.825-1.476-0.5680.8661.2281.756

-0.888-0.475-0.894-1.851-0.140-0.618-2.0381.3471.8611.9943.4344.2525.7176.0696.6394.0383.9774.5965.4136.7194.7454.8014.080

z

0.3770.8600.7221.1572.3310.2420.097

-1.0560.6301.6190.0640.8771.2130.2441.362

-0.871-1.012-0.582-0.978-2.159-0.037-0.4410.752

-1.395-1.176-0.458-0.4690.756

-1.273)

Dihedral angle

168.0-115.2

'I 98.3Xi 176.8X2 84.4

(a)

Xi

XIX2

'P

(a

The structure has been optimized for = 1200 and d = 1.65 A(0120dl.65). Each successive tetrapeptide unit may be generated bya z-axis rotation of -120° and a rise of -1.65 A.

approximate triangle, with alternate asparagines at each apexand the interior filled with the remaining asparagine sidechains. There is an open, nonideal type II ,3 turn at each ofthe apical asparagine residues. Alanine and proline residuesprovide a hydrophobic central region for each face. Fig. 3presents a schematic diagram of the hydrogen bondingnetwork for this structure. The five strong helix-stabilizinghydrogen bonds joining proximal tetrapeptides range inlength from 1.91 to 2.19 A and are shown with dashes. Thefour weaker intratetrapeptide hydrogen bonds of each unit,drawn with dots, are generally longer (2.08-2.66 A) and areless linear.The possibility was then considered that 0120dl.65 and

8170d2.8 might not be stable if the helical constraints wererelaxed. To this end, two chains of 60 residues each wereconstructed and subjected to molecular dynamics simula-tions at 300 K without symmetry constraints. After 15 ps thehelical chain derived from 0120dl.65 was virtually un-changed. However, after 15 ps the ends of the ribbon-likestructure (0170d2.8) were considerably frayed, and after 44 psa bulge appeared in the center. These results indicate that6170d2.8 is probably not stable in the absence of explicithelical constraints.The 0120dl.65 structure was also subjected to conforma-

tional changes within the overall constraints of the helicalparameters. For example, a concerted flip of the asparagineside chains could arise if a slightly different starting confor-mation had been used. This would not change the number ofhydrogen bonds or the nature of the helix but could result ina lower energy. Therefore, starting with 0120dl.65, the 128possible combinations of 00 and 1800 rotations of the side-

chain torsion angles and main-chain peptide plane rotationsand cis/trans proline rotations were generated. Simulationsat 1000 K and 3000 K were also carried out to induceadditional transitions. Several of the variants were almost asenergetically favorable as the initial 0120dl.65 structure(within 3 kcal/mol of tetramer). These structures were notanalyzed in detail, however, as each retained the right-hand-ed 1238 helical motif shown in Fig. 1.

DISCUSSIONSome caution must be exercised in attempting to relate the1238 helix shown in Fig. 1 either to the native structure of thePf CS protein repeat region or to the structure of variousfragments in solution. Most ofthe calculations assumed somesort of helical symmetry about a repeat of four amino acidsand solvent was not explicitly included. It may be argued thatonly a small fraction of the possible templates was tried.Furthermore, it is possible that the repeating region of the CSprotein has no organized structure, as suggested by thepresence of asparagine and proline, both of which tend tobreak a-helical and -sheet structures (26). In such a case, apossible function of the repeat region of the CS protein mightbe to present a random surface on the sporozoite for thepurpose of immune system evasion by stimulating an irrele-vant immune response (16). However, this view is probablynot correct since antibodies to the repeats prevent thedevelopment of the parasite (29).Two additional factors also lend support to our approach

and to the final structure itself. First, since the central regionofthis malarial CS protein has evolved as an unusual repeatedtetrapeptide and is conserved in many strains of Pf (30), it islikely to have a stable, repetitious structure. Second, resultsof circular dichroism and infrared spectral studies on oligo-mers of Asn-Pro-Asn-Ala suggest that the 60-mer has asignificant amount of secondary structure similar to /3 turnsand is more highly ordered than the 40-mer (T. J. O'Leary,personal communication). Such an observation might beexpected for a helix consisting of a relatively large number ofamino acids per turn. In the case of the 40-mer, only threecomplete turns of the 1238 helix are possible, whereas the60-mer could incorporate five full turns ofthe helix, providinga reasonable degree of stabilization.

CONCLUSIONSTesting of potential malarial vaccine components containingAsn-Pro-Asn-Ala repeats in humans is already underway (29,31). Therefore, a theoretical understanding of oligomers ofthis peptide is of timely interest. The results presented herehave led us to three conclusions regarding the design ofvaccine components. These are based on the assumption thatthe native structure is important for induction of a protectiveimmune response.

First, it is likely that more than three turns are required toimpart stability to the 1238 helix. Consequently, the modelpredicts that the repeating regions of Pf CS vaccines shouldbe >36 residues in length to mimic the native conformation.

Second, it may be seen in Fig. 2 that the 0120dl.65structure predicts that the variant regions ofthe PfCS proteinhaving the sequence Asn-Val-Asp-Pro would have theaspartic acid side chains turned inward toward the centralhelical axis. This may be unfavorable and a similar structurewith the side chain of the residue preceding each proline onthe outside may be preferred. However, for all of thesestructures, it is expected that the ionized carboxylate groupwould hydrogen bond with the NH2 groups of the asparagineside chains in the adjacent turns ofthe helix, thus ordering theentire hydrogen bond array in a specific manner. Since thefirst three of the Val-Asp replacements are separated by eight

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FIG. 1. Stereo side view of the most favorable calculated structure for an oligomer of the Pf CS protein repeating tetrapeptide having 15Asn-Pro-Asn-Ala units. The helical axis is aligned vertically with the N terminus at the bottom. The structure has been optimized for 9 = 1200and d = 1.65 A (0120dl.65). Five turns of the helix, which has 12 amino acids per turn and a pitch of 4.95 A, are shown. Hydrogens attachedto carbon are omitted. Color coding: yellow, carbon; white, hydrogen attached to nitrogen; blue, nitrogen; red, oxygen.

residues, they will not conflict with each other in that eachwill be part of a separate vertical hydrogen-bonding network.This implies that the Val-Asp replacements might be essentialin the selection of the native structure and should be includedin vaccine components.

Third, the results of this work also indicate that there is aclass of almost isoenergetic 1238 helical structures. Thesestructures can be generated from the 0120dl.65 structure byrotating some peptide planes or side-chain torsions, or evenchanging the helix sense from right-handed to left-handed(i.e., to the analogous 0120dl.65 structure). For all of thesestructures the stabilization is provided by similar hydrogen-bonding networks. These structures, though all possible in anisolated repeating peptide, would not all be able to accommo-date the presence of the Val-Asp replacements or the tem-

plate provided by the rest of the CS protein. It is for thesereasons that we believe that a trial vaccine should include therepeating portion of the CS protein and some of the lead-insequence adjacent to the repeat portion to aid in the selectionof the correct isoenergetic helical structure.

After completion of this study, a prediction ofthe structureof the repeating unit (Asn-Ala-Asn-Pro)6 by Gibson andScheraga appeared (32). Using a modified build-up procedure(23) for their conformational search, they arrived at twolow-energy structures: an extended, left-handed helix of 6residues per turn with a pitch of 6.96 A and a more compact,right-handed helix of 12 residues per turn with a pitch of 9.91

. The latter was proposed to be the native structure. Thestructures found by Gibson and Scheraga are fundamentallydifferent from those presented in this paper. Though0120dl.65 is similar to their compact helix in that it also has12 residues per turn, the pitch of 0120dl.65 is only 4.95 A andit is extensively stabilized by hydrogen bonds between atomsseparated by 12 residues along the chain. No stabilizinghydrogen bonds are apparent in the Gibson-Scheraga struc-

H .....j.....H1 13 14 15

H1 16

-c-N-CH-C-'--N-CH-C-N-CH-C-N-CH-C-I I IU / \ N I 11 I 11o CH2 0° CH2 .° CH3 0

: +\ : .:| N-. H-N"'\ l1

I I * . :1I | * H|eee-@- @ :1@I : I I I::

I H | H

NH4CNC

-CCH--CNCH-C-

CH2 1° 0 H2 CH3 0

0 -CNH H-N"40I

H H

FIG. 2. End view down the helical axis of the most favorable

calculated structure (8120dl.65) for an oligomer of the Pf CS proteinrepeating tetrapeptide having nine Asn-Pro-Asn-Ala units. The threeAsn-Pro-Asn-Ala tetramers in one turn are shown. Helical parame-ters and coding conventions are as in Fig. 1.

FIG. 3. Schematic diagram of the hydrogen-bonding network inthe most favorable calculated structure (0120dl.65) for oligomers ofthe PfCS protein repeating tetrapeptide shown in Figs. 1and 2.-,Strong interpeptide H bonds between Asn-Pro-Asn-Ala units inadjacent rungs of the helix.., Weaker intrapeptide H bonds.Intrapeptide H bonds in the lower unit and those H bonds extendingabove and below the figure have been omitted for clarity.

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ture. In fact, their structure most closely resembles 6120d3.3,the energy of which is =20 kcal/mol of tetramer higher than0120dl.65.

We are grateful to Drs. R. L. Beaudoin and R. Wistar, Jr., of theNaval Medical Research Institute, to Drs. C. L. Diggs and W. T.Hockmeyer of the Walter Reed Army Institute of Research, and toDr. D. T. Liu of the Food and Drug Administration for encouragingus to pursue this investigation. We also thank Dr. L. H. Miller of theNational Institutes of Health for providing the Pf CS proteinsequence in advance of publication. Dr. T. J. O'Leary of the Foodand Drug Administration kindly discussed his circular dichroismresults with us prior to their publication. Helpful discussions withDrs. R. J. Feldmann, B. K. Lee, and D. Lipman of the NationalInstitutes of Health speeded the progress of this study. Additionalthanks are due to Dr. M. Karplus of Harvard University for readingthe manuscript and providing helpful comments. F.W.C. was sup-ported by the Office of Naval Research through Summer FacultyResearch Associateships and as a Visiting Scientist through anIntergovernmental Personnel Act assignment to the Naval MedicalResearch Institute. This work was supported in part by NavalMedical Research and Development Command Work Unit3M162770A870AF312.

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