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Role of the N-terminal part of the coat protein in the assembly of cowpea chlorotic mottle virus
J. Mol. Biol. (1986) 191, 453-460
Role of the N-terminal Part of the Coat Protein in the Assembly of Cowpea Chlorotic Mottle Virus A 500 MHz Proton Nuclear Magnetic Resonance Study and Structural Calculations G. Vriend19 7, B. J. M. Verduin2 and M. A. Hemmingal’
$
’ Department of Molecular Physics, Agricultural University de Dregen 11, 6703 BC Wageningen ’ Department of Virology, Agricultural University Binnenhaven 11, 6709 PD Wageningen, The Netherlands (Received 14 November 1985, and in revised form 19 May 1986) The interaction of the oligonucleotides (Ap)sA and (A-T), with empty capsids of the coat protein of cowpea chlorotic mottle virus (CCMV) has been studied with 506 MHz ‘H nuclear magnetic resonance. It is found that these oligonucleotides specifically bind to the arginine and lysine residues of the N-terminal arm of the protein. Upon this binding, immobilization of part of the N-terminal arm occurs. In addition, secondary structure predictions and energy calculations have been performed on the N-terminal arm. These calculations were carried out as a function of the charges on the arginine and lysine side-chains. For free coat protein, where the arginine and lysine side-chains are charged, the arm is found in a random-coil conformation. In the neutralized state, as for the coat protein in the virus, the arm adopts an m-helical conformation. The results support a previously published model for the assembly of CCMV, in which a random-coil to u-helix conformational transition, induced by neutralizing the arginine and lysine side-chains, plays an essential role.
1. Introduction Cowpea chlorotic mottle virus (CCMQ) is a spherical plant virus of the group of bromoviruses. It consists of RNA surrounded by 180 identical, icosahedrally arranged protein subunits (Bancroft et al., 1967). The molecular mass of a virus particle is approximately 4.6 x lo6 daltons. The virus is stable around pH 5.0 (Bancroft & Hiebert, 1967). When the pH is raised to 7.5 at high ionic strength (I > 0.3). the virus dissociates into protein dimers
t Present address: Laboratory of Chemical Physics, University of Groningen, Nijenborgh 16, 9747 AG Groningen. The Netherlands. $ Author to whom all correspondence should be addressed. 4 Abbreviations used: CCMV, cowpea chlorotic mottle virus; n.m.r., nuclear magnetic resonance; (AI))~A, ribonucleic acid form of (adenosine-phospho),-adenosine; (A-T),, deoxyribonucleic acid form of (adenosinephospho-thyminephospho),-adenosine-phospho-thymine; p.p.m., parts per million: T. triangulation number; D, deuterinm. 002%2836/86/190453-08
$03.00/O
453
and RNA almost free of protein (Bancroft & Hiebert, 1967). Both RNA and protein can be isolated and reassembled in vitro (Bancroft & Hiebert, 1967). In the absence of RNA, the protein subunits reassociate into empty protein capsids by lowering the pH to 5-O (Bancroft et al., 1968). These empty protein capsids have a structure similar to the native virus: a T = 3 icosahedral surface lattice (Finch & Bancroft, 1968). The protein subunit contains a basic N-terminal arm of 25 amino acid residues that can be cleaved by tryptic digestion (Chidlow & Tremaine, 1971). This arm has been shown to be the RNA-binding part of the protein (Vriend et al., 1981). The N-terminal arm contains six arginine, three lysine and no acidic residues (Rees & Short, 1982; Dasgupta & Kaesberg, 1982). It has been suggested that interactions between the positive side-chains of these amino acids and the negative phosphate groups in the RNA are responsible for the proteinRNA interaction (Vriend et al., 1981; Argos, 1981). The assembly process in vivo is very specific. In vitro, however, no specificity has been observed and CCMV protein tends to associate with any poly0
1986
Academic
Press
Inc.
(London)
Ltd.
G. Vriend
Stage
et
al.
I
Stage
3
o-Helix I
Mobile
arm
CCMV-protein
Figure 1. “Snatch-pull’ interaction model of binding of CCMV protein to RNA (Vriend et al., 1982; Hemminga et al.. 1985). In stage 1, electrostatic interaction takes place between mobile positively charged protein arms and the negatively charged phosphate groups of the RNA. In stage 2, the protein arm has snatched the RNA and neutralization
of chargestakes place. In stage3, the arm rolls up into an a-helix and pulls the protein to the RNA. In solution, CCMV protein-exists
mostly
as
dime&
nucleotide, or even polyanions like polyvinyl sulphate or sodium dextran sulphate (Bancroft et al., 1969). From n.m.r. measurements, it has been found that the N-terminal arm is very mobile in the absence of RNA, whereas immobilization occurs upon binding RNA (Vriend et al., 1981). Twodimensional nuclear Overhauser effect n.m.r. spectroscopy experiments have revealed that the N-terminal arm is for the major part in a randomcoil conformation in the absence of RNA (Vriend et al., 1985). A “snatch-pull” model has been proposed for the assembly of CCMV protein and RNA (Vriend et al., 1982; Hemminga et aZ., 1985). In this model (see Fig. 1) the N-terminal part of the protein is considered a mobile arm attached to a relatively rigid protein body. The arm is in a flexible random-coil conformation before the interaction takes place. This flexibility enhances the chance of arginine and lysine side-chains to meet with phosphate groups in the RNA. Upon binding, positive arginine and lysine residues, and negative phosphate groups neutralize each other, and the arm rolls up into a rigid a-helix, thereby pulling the protein body to the RNA. Experimental evidence has been provided for the first two steps in the model. However, little is known about the structure of the N-terminal arm when bound to the RNA. Laser Raman studies have shown that the N-terminal arm in CCMV has a regular structure, but no conclusions have been drawn about the nature of this structure (Verduin et al., 1984). High-resolution X-ray structures have been obtained for four spherical RNA viruses: tomato bushy stunt virus (Harrison et al., 1978), southern bean mosaic virus (Abad-Zapatero et al., 1980), satellite of tobacco necrosis virus (Liljas et al., 1982) and human common cold virus R14 (Rossman et al., 1985). These viruses have a quaternary structure similar to CCMV. However, in these structures most of the N-terminal protein arms cannot be observed, or only partly, because they are bound to RNA with several non-equivalent local structures. Therefore, it seems unlikely that
the complete structure of the N-terminal arm of CCMV can be obtained by X-ray diffraction techniques. Also, it is not possible to elucidate the structure of the N-terminal arm with highresolution n.m.r. because too strongly broadened spectra are obtained of intact virus (Vriend et al., 1981), or complexes of CCMV protein with oligonucleotides (Hemminga et al., 1985). Also, solid-state n.m.r. techniques are not yet well enough developed to provide such det’ailed information (Hemminga et al., 1985). The objective of this study was to obtain information about the protein-nucleic acid interactions in CCMV, by studying with ‘H n.m.r. the binding of the oligonucleotides (Ap)sA and (A-T), to empty capsids of CCMV coat protein. From these binding experiments, it is concluded that interactions occur between the oligonucleotides and arginine and lysine residues of the N-terminal arm. The part of the N-terminal protein arm that primarily interacts with the oligonucleotides is found to range from approximately residue 10 to 25. Furthermore, evidence is provided for the third step in the “snatch-pull” model for the proteinRNA interaction (Fig. 1) by structure predictions and energy calculations.
2. Materials (a) Preparation
and Methods of viral
coat protein
Virus was purified as described (Verduin, 1978). Coat protein prepared from virus by the CaCl, method (Verduin, 1974) was dialysed against 300 mM-NaCl, 10 m&i-MgCl, and 10 mM-sodium phosphate (pH 5.0). H,O in this solution was substituted by D,O by extensive dialysis against the above solution made up in D,O. In D,O, pH meter readings were taken without correction for the presence of D,O. The protein concentration was initially 2.0 mg/ml. To obtain protein lacking the N-terminal arm. coat, protein was dialysed against 300 mM-NaCl, 50 mMTris . HCl (pH 7.5) and treated for 24 h at 5°C with 5 mg of Trypsin 30 Enzygel (Boehringer) per 100 mg of protein. After incubation, the gel was removed by lowspeed centrifugation and the supernatant was dialysed
Role of the N-terminal
Arm in CCM V Assembly
455
Protem:(Ap),A=l.ZO w
I
I
1
I
I
I
IO
I
1
1
I
5 p.p.m.
I
I
1
0
Figure 4. 500 MHz ‘H n.m.r. spectra of the ribonucleotide (Ap),A (trace A) and the deoxyribonucleotide (A-T), (trace B) at pH 5.0. Protein
(c) Nuclear magnetic I IO
I
,
I
,I
I 5
,,,I, 0
P.P.m
Figure 2. 500 MHz ‘H n.m.r. spectra of titration of (Ap),A to empty capsids of CCMV coat protein at pH 5.0. The indicated ratios are between protein subunits and oligonucleotides on a monomer basis. The protein concentration is 0.25 pmol/ml. The inset shows the lowfield region of the ‘H n.m.r. spectrum of 0.25 pmol (Ap),A/ml solution at pH 5.0. against 1 M-NaCl, 50 mw-sodium acetate (pH 5.0) to remove the small cleavage products and induce the formation of empty casids. (b) Oligonucleotides The oligoribonucleotide (Ap)sA was obtained from Boehringer-Mannheim GmbH. (A-T), (deoxy form) was a kind gift from Professor J. H. van Boom.
(A-T),=1
I.5
Protein
(A-T15=I
0.6
Protein.
(A-T),
measurements
‘H n.m.r. spectra were recorded with a Bruker WM500 spectrometer. Samples of 400 to 600 ~1 were measured at 7°C in the quadrature detection mode with D,O lock. The acquisition time was 0.4 s and 2048 scans were taken. The sensitivity enhancement was 5 Hz. The p.p.m. scale was relative to sodium 2,2-dimethyl 2-silapentane-5sulphonate. The vertical scale was corrected for concentration differences between the samples of each series of experiments. The HDO peak was presaturated for 2.0 s with 1.8 W decoupling power. The samples were dissolved in D,O containing 300 mM-NaCl. 10 mM-MgCl, and 10 mM-sodium phosphate at pH 5.0. (d) Secondary
structure
predictions
Predictions were performed using the methods of Chou & Fasman (1974a,b), Burgess et al. (1974) and Lim (1974a,b). The programs were originally written by Dr J. A. Lenstra, and adapted by us for interactive use at a DEC 10 computer. (e) Energy
Protein
resonance
calculations
Calculations were carried out using the program UNICEPP, coupled to a minimization procedure according to Powell (1964). These programs were obtained through the Quantum Chemistry Program Exchange library from the Chemistry Department of the Indiana University (Indiana. U.S.A.). The principles of the program UNICEPP have been described extensively (Momany et al., 1970, 1971, 1975; Yan et al., 1970: McGuire et al., 1972; Lewis et al., 1973: Dunfield et al., 1975). Initial structures were generated, and final structures evaluated on a Vector General picture system, using the program MANIPX, which was originally developed at the Texas A&M University.
= I : 0.0
3. Results and Discussion (a) Nuclear magnetic resonanceexperiments Figure 3. 500 MHZ ‘H n.m.r. spectra of titration of (A-T), to empty capsids of CCMV coat protein at pH 5.0. The indicated ratios are between protein subunits and oligonucleotides on a monomer basis. The protein concentration is 0.25 /Imol/ml. The inset shows the lowfield region of the ‘H n.m.r. spectrum of a 0.25 pmol (A-T)5/ml solution.
The ‘H n.m.r. spectra in Figures 2 and 3 show the effect of addition of (Ap),A and (A-T),, respectively, to empty protein capsids at pH 5-O. For reference purposes, the ‘H n.m.r. spectra of (Ap),A and (A-T), at pH 5-O are given in Figure 4. Figure 2 trace A represents the n.m.r. spectrum of the mobile N-terminal arm in empty protein
456 5 Ac-Ser-Thr-Val-Gly-Thr-Gly-Lys-Leu-Thr-Arg-Ala-Gln-Arg-~g-
15 20 Ala-Ala-Alo-Arg-Lys-Asn-Lys-Arg-Asn-Thr-Arg-
G. Vriend et al IO
IMethodISTVGTGKLTRAQRRAAARKNKRNTR
1
25
Figure 5. Primary structure of the N-terminal the CCMV protein (Dasgupta & Kaesberg, 1982).
arm of
I I C+F (mod.) LIM
w Helix
capsids only. The n.m.r. signals of the rest of the protein are broadened beyond detection (Vriend et al., 1981). From the spectra in Figure 2 traces B to D it can be estimated that approximately one molecule of (Ap),A binds to one N-terminal arm. This calculation is based on monomers of coat protein and (Ap),A. Actually, (Ap),A is present as a double-stranded structure (Hilbers, 1979; Olsthoorn et al., 1981), and coat protein as empty capsids assembled before oligonucleotide binding. A striking observation in Figure 2 trace D is that the Arg C6H resonance at 3.2 p.p.m. is almost completely broadened, whereas approximately 30% of the intensity of the Lys C”H resonance at 3.0 p.p.m. remains observable. This indicates that not the whole of the N-terminal arm is immobilized, only part of it. Knowing the amino acid sequence of the N-terminal arm (see Fig. 5), this indication appears to be consistent with the idea that the amino acid residues 10 to 25 (containing all the arginine and 2 of the 3 lysine residues) immobilize upon binding of (Ap),A. This idea is supported by the fact that other proton resonances of the first seven amino acid residues appear as sharp peaks in the spectrum in Figure 2 trace D, i.e. Val CYH (-l.Op.p.m.), Thr CYH (~1.2p.p.m.), Lys CYH (N 1.4 p.p.m.), acetyl group of Serl (-2.1 p.p.m.). The high-field shoulder of the Val CYH resonance at - 1.0 p.p.m. is identified as Leu C6H (Vriend et al., 1985). However, this signal is not sufficiently resolved to decide whether Leu8 remains mobile upon addition of (Ap)sA, or not. Therefore, no definite conclusions can be drawn about the state of the amino acid residues Leu8 and Thr9. Figure 3 shows the result of the addition of double-stranded (A-T), to empty protein capsids at pH 5.0. Although many protein peaks in the 1 to 5 p.p.m. region overlap with (A-T), peaks, it can be seen that the same amino acid residues remain mobile as is observed for the titration with (Ap)sA. In this case also, approximately one molecule of (A-T), binds to one N-terminal arm (based on monomers). As a control experiment, addition of (Ap),A and (A-T), to protein lacking the N-terminal arm, in the form of empty capsids at pH 5.0, has been studied by n.m.r. spectroscopy. From the fact that no effect on the n.m.r. signal of the oligonucleotides is observed, it is concluded that these oligonucleotides bind only to the N-terminal arm. From the n.m.r. experiments, it is clear that the N-terminal arm of CCMV protein is necessary for oligonucleotide binding. The fact that the oligonucleotides (Ap),A and (A-T), can bind to the
m
Sheet
/vv\
Turn
JJj,
Figure 6. Secondary structure predictions for the E-terminal arm of CCMV protein. C+F, the method of Chou & Fasman (1974aJ). B et al., the method of Burgess et al. (1974). LIM, the method of Lim (1974uJ). C+F (mod.), the method of Chou & Fasman (1974aJ) after lowering the a-helix potentials for the arginine and lysine side-chains by 25%. Predictions C+F and B et al. simulabe neutral arginine and lysine side-chains. Predictions C+F (mod.) and LIM simulate charged sidechains.
empty capsids suggests that the N-terminal arms are accessible from the outside of the capsid. This is in agreement with binding studies of CCMV RNA to empty capsids (Bancroft, 1970). No significant, differences in binding characteristics (stoichiometry, immobilization, amino acids involved) are found for (Ap),A and (A-T),. This indicates that in vitro there is no specificity for deoxyor ribonucleotides or for the base sequences. This is in good agreement with other results (Bancroft et al.. 1969). (b) Secondary
structure productions
Figure 6 shows the result of the secondary structure prediction for the N-terminal arm of the coat protein of CCMV, using the method of Chou & Fasman (1974aJ) and the result using the method of Burgess et aE. (1974). It is seen that an a-helix is predicted from approximately residue 10 to 20. This prediction is in good agreement with that for the coat protein of another bromovirus, brome mosaic virus (Argos, 1981). This is not surprising, since the primary structure of the N-terminal arm of this virus protein and that of CCMV protein are quite similar. Also the N-terminal arm of the coat protein of brome mosaic virus has been found to immobilize upon binding RNA (Vriend et al., 1982). In Figure 7, a double a-helical net representation (Lim? 1978) of the N-terminal arm is given. This shows that an a-helical fold would bring all positive charges in close spatial proximity, which is very unlikely to occur. The fact that an a-helix is nevertheless predicted is readily explained from the nature of these two prediction methods. Both calculate from a small set of known structures the probabilities that amino acids are found in helix, sheet, turn or random-coil conformation. The probabilities, or structure potentials, are then applied to the amino acid sequence for which the secondary structure has to be predicted. The datasets used to derive the structure potentials,
Role of the N-terminal Arm
in CCM V Assembly
457
(c) Energy N
N
R
T
R
A L
G
' S
T
v
G
' S
v T
Figure 7. A double cc-helicalnet representation (Lim, 1978)of the N-terminal arm of CCMV. In this net, 2 arms are placed next to each other, to enablethe observation of the a-helical conformation from all directions. The distancebetweenadjacent residuesis proportional to the distancebetweentheir C” atoms in an a-helix. One region containing all arginine (R) and lysine (K) amino acid residuesis hatched.
however, contain almost no clusters of charged residues. Actually, many arginine and lysine residues in these datasets are in a neutralized state. Therefore, these predictions correspond to the case in which the positive charges in the N-terminal arm are neutralized, as in the native virus. To simulate the effect of repulsive forces between the positive charges in the N-terminal arm, the a-helix potentials for arginine and lysine in the Chou & Fasman method (Chou & Fasman, 1974a) were lowered by 25%. The corresponding prediction is shown in Figure 6, as well as the result of the prediction using the method of Lim (1974a,b). The latter method is not based on statistics, but employs general principles of protein folding and stability. It explicitly treats arginine and lysine side-chains as large hydrophilic, for which quite a number of u-helix prohibition rules apply. These predictions clearly demonstrate a random-coil instead of the a-helix for the case in which arginine and lysine side-chains are charged, i.e. for the IV-terminal arm of the coat protein of CCMV free in solution. Argos et al. (1978) have shown that the a-helix parameters for arginine and lysine can change by approximately 20% by changing the database. This means that a decrease of 25% as taken in the modified Chou & Fasman method (C+F (mod.) in Fig. 6) is well possible. It is also found that the probability for the formation of an u-helix in the region from residue 10 to 20 rapidly diminishes on decreasing the a-helix potentials of arginine and lysine, leaving definitely no a-helix left at a decrease of 25%.
calculations
The secondary structure predictions indicate that the charges on the arginine and lysine side-chains determine the conformation of the N-terminal protein arm. To check this, energy calculations were performed as a function of these charges. Because the computational task required to perform even a limited grid search over the conformational space of the 25residue long N-terminal arm is far too large for the current generation of computers, we could calculate only the relative energy contents of a small number of structures. From laser Raman studies, it is known that some secondary structure is present in the N-terminal protein arm in the native virus (Verduin et al., 1984). Therefore, it was decided to take two starting conformations in the a-helix region, and three in the b-sheet region. In these starting conformations, all amino acid residues had the same dihedral angles. The starting values for these dihedral angles are indicated by a cross in the Ramachandran plots given in Figure 8. From these starting conformations, energy minimizations proceeded for three different values of the partial charge p on the arginine and lysine side-chains. The dihedral angles of the residues in the structures obtained after the energy minimization are indicated in these Ramachandran plots. Table 1 shows the relative energy contents for these final structures. It can be seen that the u-helix conformation is energetically more preferable than the P-sheet when the arginine and lysine side-chains are neutralized (p = 0), simulating the situation in the virus, where these side-chains have a strong electrostatic interaction with phosphate groups of the RNA. In the absence of RNA, arginine and lysine side-chains in the N-terminal protein arm are not neutralized, or only partly by counter ions in the solutions. These two situations are simulated by p = 1.0 and 0.5, respectively. Although the a-helix remains the favourite structure with fully charged arginine and lysine side-chains, it is clearly seen that t’he energy differences between the several structures become less significant with increasing charges. This implies Table 1 Minimized energy content of some u-helix and p-sheet conformations of the N-terminal part of CCM V protein as a function of the partial charge (p) on the side-chains of the arginine and lysine-residues
Startingvalues Conformation LX1 a11
1 cal=4.184J.
Energycontent(kcal/molt) relative
~~
to an arbitrary reference point -~~~~~~~~ p = 0.5 p=
4
i
~~~~ p=O
-57 - 50
-57 -50
-227 -238
-126 - 127
63 51
-188 -169 -155
-71 -80 -71
87 66 67
-30 -50 -150
;:I /?I11 t
of the dihedral angles (deg.) ~-
150 150 150
1.0
c
”
a1 (I.01
a1 (01
a1 (0.5)
k$4=(-52-57)
b$,W=(-57,-57)
@,qJ)=(-52-57)
E=-227
E=-126
E=63
i 0 -
e
0
aII(O)
aII(O.5)
aII(1.0)
MqJI=(-50,-50)
(+,+1=(-50.-50)
(~,+)=(-50,-50)
E=-127
E=51
P
I
I
E=-238
T
c PI(O.5)
DI (I.01
(4?44=(-30.150)
k$,$JH-30350)
E=-71
E=87
p1r (01 NyJJ)=~-5o,l50) E=-169
I
0
pII1 (1.0) ~4!4J)=(-l5o,l50) V
E=67 -180
0
180 9---
Role of the N-terminal
Arm
CCMV
Assembly
459
has been suggested that double-stranded nucleotide helices are required for binding to the N-terminal arm of CCMV protein (Hemminga et al., 1985).
that the N-terminal arm is mostly in a random-coil conformation in solution, in accordance with the secondary structure predictions. This observation does not result from the fact that different local minima were obtained in the presence or absence of charges. This was checked by toggling p between 0 and 1.0 after completing the minimization and of the minimization subsequent continuation procedure. The values for the energy content obtained in this way were only slightly larger (i.e. up to +3I kcal/mol for fir with p changed from 1.0 to 0 (1 cal = 4.184 J)) than the values given in Table 1. From Figure 8 it can be seen that, after energy minimization, the residues 1 to 7 often have dihedral angles strongly deviating from the rest of the peptide chain. This probably arises from the presence of glycine residues at positions 4 and 6. It is known that glycine can cause structural irregularities (Lim, 1974a,b). From the calculations it is found that 75% of the differences in energy content in Table 1 is determined by electrostatic interactions, the remaining 25% arising from non-bonded and torsion interactions. This means that the electrostatic terms in the calculations are the dominating factors for the conformational changes, justifying our approach.
4. General
in
The authors are indebted to ProfessorJ. H. van Boom for providing (A-T),. We gratefully acknowledge the technical assistance of Miss H. Bloksma with the virus preparation. The authors thank the staff of the Computer Center for their continuous support during the many hundreds of hours CPU time needed for this work, and Miss M. Gerritsen for her assistance with the installation of the UNICEPP program package and the energy minimization calculations. We enjoyed stimulating discussions with Professor T. J. Schaafsma. This research was supported by the Netherlands Foundation for Biophysics, with financial aid from the Netherlands Organization for the Advancement of Pure Research (ZWO). The 500MHz ‘H n.m.r. investigations were supported by the Netherlands Foundation for Chemical Research (SON) with financial aid from ZWO.
References Abad-Zapatero, C., Abdel-Meguid, S. S., Johnson, J. E., Leslie, A. G. W., Rayment, I.. Rossman, M. G., Suck, D. & Tsukihara, T. (1980). &&re (London), 286, 33-39. Argos, P. (1981). Virology, 110, 55-62. Argos, P., Hanei, M. & Garavito, R. M. (1978). FEBS Letters, 93, 19-24. Bancroft, J. B. (1970). Advan. Virus Res. 16. 99-134. Bancroft, J. B. & Hiebert, E. (1967). VimoZogy, 32, 354356. Bancroft, J. B., Hills, G. J. & Markham, R. (1967). Virology, 31, 354-379. Bancroft, J. B.. Wagner, G. W. & Bracker. C. E. (1968). Virology, 36, 146-149. Bancroft, J. B., Hiebert, E. & Bracker, (‘. E. (1969). Virology, 39, 924-930. Burgess, A. W., Ponnuswamy, P. K. & Scheraga, H. A. (1974). Isr. J. Chem. 12. 239-286. Chidlow, J. & Tremaine, J. H. (1971). J’irology, 43, 267278. Chou, P. Y. & Fasman, G. D. (1974a). Biochemistry, 13, 211-221. Chou. P. Y. & Fasman, G. D. (19746). Biochemistry, 13, 222-245. Dasgupta, R. C Kaesberg, P. (1982). ;Vucl. Acids Res. 10, 703-713. Dunfield, N., Burgess, A. W. & Scheraga. H. A. (1975). J. Phys. Chem. 82, 2609-2616. Finch, J. T. & Bancroft, J. B. (1968). Nature (London), 220, 815-816. Harrison, S. C., Olson, A. J.. Schutt, C. E., Winker, K. K. & Bricogne, G. (1978). Nature (London), 276, 36% 373. Hemminga, M. A., Datema, K. P.. Ten Kortenaar, P. W. B.. Kriise, J., Vriend, G.. Verduin, B. J. M. & Koole, P. (1985). In Magnetic Re,sonance in Biology and Medicine (Govil, G., Khetrapal. C. L. & Saran, A., eds), pp. 53-76, Tata McGraw-Hill. New Delhi.
Discussion
Binding of (Ap)sA or (A-T), to empty protein capsids results in immobilization of the amino acid residues 10 to 25 in the N-terminal arm. Secondary st,ructure predictions and energy calculations have indicated that, upon neutralizing the positively charged arginine and lysine side-chains of the N-terminal arm, an a-helix is formed between residues 10 and 20. This corresponds well to the region that is immobilized, suggesting that an m-helix is formed upon binding the oligonucleotides. Similar a-helix conformation may occur in native virus and, if so, the suggested snatch-pull interaction model may be a representation of the assembly model in viva (seeFig. 1). The fact that in the predictions the amino acid residues 21 to 25 of the N-terminal arm in empty protein capsids do not form an a-helix but immobilize in the n.m.r. experiments, may be explained by noting that the N-terminal arm is fixed to protein “bodies” assembled in a capsid that performs a slow rotational motion. In the a-helix, all arginine and lysine side-chains are located at one side, facilitating binding to a regular array of negat
Figure 8. Ramachandran plots of the u-helix (aI( aII(p)) and b-sheet (PI(p), pII( pIII(p)) different values of the partial charge p. X, Starting point of the conformations with dihedral angles angles after energy minimization. Amino acid residues whose dihedral angles deviate significantly values are indicated by name. In most cases, these residues are in the region from residue 1 to 7. The rontent E is in kcal/mol relative to an arbitrary reference point (see Table 1).
conformations at (4, II/). 0, Dihedral from the starting minimized energy
G. Vriend et al. Hilbers,
C. W.
(1979).
In
BioZogicaE Appkations of R. G., ed.), pp. l-43, Academic Press, New York. Lewis, P. N., Momany, F. A. & Scheraga, H. A. (1973). fsr. J. Chem. 11, 121-152. Liljas, L., Unge, T., Jones, T. A., Fridborg, K., Lovgren, S., Skoglund, U. & Strandberg, B. (1982). J. Mol. BioZ. 159, 93-108. Lim, V. I. (1974a). J. Mol. BioZ. 88, 857-872. Lim, V. I. (1974b). J. Mol. BioZ. 88, 873-894. Lim, V. I. (1978). FEBS Letters, 89, 10-14. McGuire, R. F., Momany, F. A. & Scherage, H. A. (1972). J. Phys. Chem. 76, 375-393. Momany, F. A., McGuire, R. F., Yan, J. F. & Scheraga, H. A. (1970). J. Phys. Chem. 74, 2424-2438. Momany, F. A., McGuire, R. F., Yan, J. F. t Scheraga, H. A. (1971). J. Phys. Chem. 75, 2286-2297. Momany, F. A., McGuire, R. F., Burgess, A. W. & Scheraga, H. A. (1975). J. Phys. Chem. 79, 23612381. Olsthoorn, C. S. M., Bostelaar, L. J., De Rooij, J. F. M., Van Boom, J. H. & Altona, C. (1981). Eur. J. Biochem. 115, 309-32 1.
Magnetic Resonance (Scbulman,
Edited
Powell, M. J. D. (1964). Comp. J. 7, 301-307. Rees, M. W. & Short, M. N. (1982). Virology, 119, Nt503. Rossmann, M. G., Arnold, E., Erickson, J. W., Frankenberger, E. A., Griffith, J. P., Hecht, H. J.. Johnson, J. C., Kamer, G., Luo, M., Mosser, A. G., Rueckert, R. R., Sherry, B. & Vriend, G. (1985). Nature (London), 317, 145-153. Verduin, B. J. M. (1974). FEBS Letters, 45, 50-54. Verduin, B. J. M. (1978). J. Gen. Viral. 39, 131-147. Verduin, B. J. M.. Prescott, B. & Thomas, G. J. *Jr (1984). Biochemistry, 23, 4301-4308. Vriend, G., Hemminga, M. A., Verduin, B. J. M., De Wit, ,J. 1~. & Schaafsma, T. J. (1981). FEBS Letters, 134, 1677171. Vriend, G., Verduin, B. J. M., Hemminga, M. A. &. Srhaafsma, T. J. (1982). FEBS Letters, 145, 49952. Vriend, G., Hemminga, M. A., Haasnoot, C. A. G., & Hilbers, C. W. (1985). J. Magn. Reson. 64, 501-505. Yan, J. F., Momany, F. A., Hoffmann, R. & Scheraga, H. A. (1970). J. Phys. Chem. 74, 420-433.
by M. F. Moody
Role of the N-terminal Part of the Coat Protein in the Assembly of Cowpea Chlorotic Mottle Virus A 500 MHz Proton Nuclear Magnetic Resonance Study and Structural Calculations G. Vriend19 7, B. J. M. Verduin2 and M. A. Hemmingal’
$
’ Department of Molecular Physics, Agricultural University de Dregen 11, 6703 BC Wageningen ’ Department of Virology, Agricultural University Binnenhaven 11, 6709 PD Wageningen, The Netherlands (Received 14 November 1985, and in revised form 19 May 1986) The interaction of the oligonucleotides (Ap)sA and (A-T), with empty capsids of the coat protein of cowpea chlorotic mottle virus (CCMV) has been studied with 506 MHz ‘H nuclear magnetic resonance. It is found that these oligonucleotides specifically bind to the arginine and lysine residues of the N-terminal arm of the protein. Upon this binding, immobilization of part of the N-terminal arm occurs. In addition, secondary structure predictions and energy calculations have been performed on the N-terminal arm. These calculations were carried out as a function of the charges on the arginine and lysine side-chains. For free coat protein, where the arginine and lysine side-chains are charged, the arm is found in a random-coil conformation. In the neutralized state, as for the coat protein in the virus, the arm adopts an m-helical conformation. The results support a previously published model for the assembly of CCMV, in which a random-coil to u-helix conformational transition, induced by neutralizing the arginine and lysine side-chains, plays an essential role.
1. Introduction Cowpea chlorotic mottle virus (CCMQ) is a spherical plant virus of the group of bromoviruses. It consists of RNA surrounded by 180 identical, icosahedrally arranged protein subunits (Bancroft et al., 1967). The molecular mass of a virus particle is approximately 4.6 x lo6 daltons. The virus is stable around pH 5.0 (Bancroft & Hiebert, 1967). When the pH is raised to 7.5 at high ionic strength (I > 0.3). the virus dissociates into protein dimers
t Present address: Laboratory of Chemical Physics, University of Groningen, Nijenborgh 16, 9747 AG Groningen. The Netherlands. $ Author to whom all correspondence should be addressed. 4 Abbreviations used: CCMV, cowpea chlorotic mottle virus; n.m.r., nuclear magnetic resonance; (AI))~A, ribonucleic acid form of (adenosine-phospho),-adenosine; (A-T),, deoxyribonucleic acid form of (adenosinephospho-thyminephospho),-adenosine-phospho-thymine; p.p.m., parts per million: T. triangulation number; D, deuterinm. 002%2836/86/190453-08
$03.00/O
453
and RNA almost free of protein (Bancroft & Hiebert, 1967). Both RNA and protein can be isolated and reassembled in vitro (Bancroft & Hiebert, 1967). In the absence of RNA, the protein subunits reassociate into empty protein capsids by lowering the pH to 5-O (Bancroft et al., 1968). These empty protein capsids have a structure similar to the native virus: a T = 3 icosahedral surface lattice (Finch & Bancroft, 1968). The protein subunit contains a basic N-terminal arm of 25 amino acid residues that can be cleaved by tryptic digestion (Chidlow & Tremaine, 1971). This arm has been shown to be the RNA-binding part of the protein (Vriend et al., 1981). The N-terminal arm contains six arginine, three lysine and no acidic residues (Rees & Short, 1982; Dasgupta & Kaesberg, 1982). It has been suggested that interactions between the positive side-chains of these amino acids and the negative phosphate groups in the RNA are responsible for the proteinRNA interaction (Vriend et al., 1981; Argos, 1981). The assembly process in vivo is very specific. In vitro, however, no specificity has been observed and CCMV protein tends to associate with any poly0
1986
Academic
Press
Inc.
(London)
Ltd.
G. Vriend
Stage
et
al.
I
Stage
3
o-Helix I
Mobile
arm
CCMV-protein
Figure 1. “Snatch-pull’ interaction model of binding of CCMV protein to RNA (Vriend et al., 1982; Hemminga et al.. 1985). In stage 1, electrostatic interaction takes place between mobile positively charged protein arms and the negatively charged phosphate groups of the RNA. In stage 2, the protein arm has snatched the RNA and neutralization
of chargestakes place. In stage3, the arm rolls up into an a-helix and pulls the protein to the RNA. In solution, CCMV protein-exists
mostly
as
dime&
nucleotide, or even polyanions like polyvinyl sulphate or sodium dextran sulphate (Bancroft et al., 1969). From n.m.r. measurements, it has been found that the N-terminal arm is very mobile in the absence of RNA, whereas immobilization occurs upon binding RNA (Vriend et al., 1981). Twodimensional nuclear Overhauser effect n.m.r. spectroscopy experiments have revealed that the N-terminal arm is for the major part in a randomcoil conformation in the absence of RNA (Vriend et al., 1985). A “snatch-pull” model has been proposed for the assembly of CCMV protein and RNA (Vriend et al., 1982; Hemminga et aZ., 1985). In this model (see Fig. 1) the N-terminal part of the protein is considered a mobile arm attached to a relatively rigid protein body. The arm is in a flexible random-coil conformation before the interaction takes place. This flexibility enhances the chance of arginine and lysine side-chains to meet with phosphate groups in the RNA. Upon binding, positive arginine and lysine residues, and negative phosphate groups neutralize each other, and the arm rolls up into a rigid a-helix, thereby pulling the protein body to the RNA. Experimental evidence has been provided for the first two steps in the model. However, little is known about the structure of the N-terminal arm when bound to the RNA. Laser Raman studies have shown that the N-terminal arm in CCMV has a regular structure, but no conclusions have been drawn about the nature of this structure (Verduin et al., 1984). High-resolution X-ray structures have been obtained for four spherical RNA viruses: tomato bushy stunt virus (Harrison et al., 1978), southern bean mosaic virus (Abad-Zapatero et al., 1980), satellite of tobacco necrosis virus (Liljas et al., 1982) and human common cold virus R14 (Rossman et al., 1985). These viruses have a quaternary structure similar to CCMV. However, in these structures most of the N-terminal protein arms cannot be observed, or only partly, because they are bound to RNA with several non-equivalent local structures. Therefore, it seems unlikely that
the complete structure of the N-terminal arm of CCMV can be obtained by X-ray diffraction techniques. Also, it is not possible to elucidate the structure of the N-terminal arm with highresolution n.m.r. because too strongly broadened spectra are obtained of intact virus (Vriend et al., 1981), or complexes of CCMV protein with oligonucleotides (Hemminga et al., 1985). Also, solid-state n.m.r. techniques are not yet well enough developed to provide such det’ailed information (Hemminga et al., 1985). The objective of this study was to obtain information about the protein-nucleic acid interactions in CCMV, by studying with ‘H n.m.r. the binding of the oligonucleotides (Ap)sA and (A-T), to empty capsids of CCMV coat protein. From these binding experiments, it is concluded that interactions occur between the oligonucleotides and arginine and lysine residues of the N-terminal arm. The part of the N-terminal protein arm that primarily interacts with the oligonucleotides is found to range from approximately residue 10 to 25. Furthermore, evidence is provided for the third step in the “snatch-pull” model for the proteinRNA interaction (Fig. 1) by structure predictions and energy calculations.
2. Materials (a) Preparation
and Methods of viral
coat protein
Virus was purified as described (Verduin, 1978). Coat protein prepared from virus by the CaCl, method (Verduin, 1974) was dialysed against 300 mM-NaCl, 10 m&i-MgCl, and 10 mM-sodium phosphate (pH 5.0). H,O in this solution was substituted by D,O by extensive dialysis against the above solution made up in D,O. In D,O, pH meter readings were taken without correction for the presence of D,O. The protein concentration was initially 2.0 mg/ml. To obtain protein lacking the N-terminal arm. coat, protein was dialysed against 300 mM-NaCl, 50 mMTris . HCl (pH 7.5) and treated for 24 h at 5°C with 5 mg of Trypsin 30 Enzygel (Boehringer) per 100 mg of protein. After incubation, the gel was removed by lowspeed centrifugation and the supernatant was dialysed
Role of the N-terminal
Arm in CCM V Assembly
455
Protem:(Ap),A=l.ZO w
I
I
1
I
I
I
IO
I
1
1
I
5 p.p.m.
I
I
1
0
Figure 4. 500 MHz ‘H n.m.r. spectra of the ribonucleotide (Ap),A (trace A) and the deoxyribonucleotide (A-T), (trace B) at pH 5.0. Protein
(c) Nuclear magnetic I IO
I
,
I
,I
I 5
,,,I, 0
P.P.m
Figure 2. 500 MHz ‘H n.m.r. spectra of titration of (Ap),A to empty capsids of CCMV coat protein at pH 5.0. The indicated ratios are between protein subunits and oligonucleotides on a monomer basis. The protein concentration is 0.25 pmol/ml. The inset shows the lowfield region of the ‘H n.m.r. spectrum of 0.25 pmol (Ap),A/ml solution at pH 5.0. against 1 M-NaCl, 50 mw-sodium acetate (pH 5.0) to remove the small cleavage products and induce the formation of empty casids. (b) Oligonucleotides The oligoribonucleotide (Ap)sA was obtained from Boehringer-Mannheim GmbH. (A-T), (deoxy form) was a kind gift from Professor J. H. van Boom.
(A-T),=1
I.5
Protein
(A-T15=I
0.6
Protein.
(A-T),
measurements
‘H n.m.r. spectra were recorded with a Bruker WM500 spectrometer. Samples of 400 to 600 ~1 were measured at 7°C in the quadrature detection mode with D,O lock. The acquisition time was 0.4 s and 2048 scans were taken. The sensitivity enhancement was 5 Hz. The p.p.m. scale was relative to sodium 2,2-dimethyl 2-silapentane-5sulphonate. The vertical scale was corrected for concentration differences between the samples of each series of experiments. The HDO peak was presaturated for 2.0 s with 1.8 W decoupling power. The samples were dissolved in D,O containing 300 mM-NaCl. 10 mM-MgCl, and 10 mM-sodium phosphate at pH 5.0. (d) Secondary
structure
predictions
Predictions were performed using the methods of Chou & Fasman (1974a,b), Burgess et al. (1974) and Lim (1974a,b). The programs were originally written by Dr J. A. Lenstra, and adapted by us for interactive use at a DEC 10 computer. (e) Energy
Protein
resonance
calculations
Calculations were carried out using the program UNICEPP, coupled to a minimization procedure according to Powell (1964). These programs were obtained through the Quantum Chemistry Program Exchange library from the Chemistry Department of the Indiana University (Indiana. U.S.A.). The principles of the program UNICEPP have been described extensively (Momany et al., 1970, 1971, 1975; Yan et al., 1970: McGuire et al., 1972; Lewis et al., 1973: Dunfield et al., 1975). Initial structures were generated, and final structures evaluated on a Vector General picture system, using the program MANIPX, which was originally developed at the Texas A&M University.
= I : 0.0
3. Results and Discussion (a) Nuclear magnetic resonanceexperiments Figure 3. 500 MHZ ‘H n.m.r. spectra of titration of (A-T), to empty capsids of CCMV coat protein at pH 5.0. The indicated ratios are between protein subunits and oligonucleotides on a monomer basis. The protein concentration is 0.25 /Imol/ml. The inset shows the lowfield region of the ‘H n.m.r. spectrum of a 0.25 pmol (A-T)5/ml solution.
The ‘H n.m.r. spectra in Figures 2 and 3 show the effect of addition of (Ap),A and (A-T),, respectively, to empty protein capsids at pH 5-O. For reference purposes, the ‘H n.m.r. spectra of (Ap),A and (A-T), at pH 5-O are given in Figure 4. Figure 2 trace A represents the n.m.r. spectrum of the mobile N-terminal arm in empty protein
456 5 Ac-Ser-Thr-Val-Gly-Thr-Gly-Lys-Leu-Thr-Arg-Ala-Gln-Arg-~g-
15 20 Ala-Ala-Alo-Arg-Lys-Asn-Lys-Arg-Asn-Thr-Arg-
G. Vriend et al IO
IMethodISTVGTGKLTRAQRRAAARKNKRNTR
1
25
Figure 5. Primary structure of the N-terminal the CCMV protein (Dasgupta & Kaesberg, 1982).
arm of
I I C+F (mod.) LIM
w Helix
capsids only. The n.m.r. signals of the rest of the protein are broadened beyond detection (Vriend et al., 1981). From the spectra in Figure 2 traces B to D it can be estimated that approximately one molecule of (Ap),A binds to one N-terminal arm. This calculation is based on monomers of coat protein and (Ap),A. Actually, (Ap),A is present as a double-stranded structure (Hilbers, 1979; Olsthoorn et al., 1981), and coat protein as empty capsids assembled before oligonucleotide binding. A striking observation in Figure 2 trace D is that the Arg C6H resonance at 3.2 p.p.m. is almost completely broadened, whereas approximately 30% of the intensity of the Lys C”H resonance at 3.0 p.p.m. remains observable. This indicates that not the whole of the N-terminal arm is immobilized, only part of it. Knowing the amino acid sequence of the N-terminal arm (see Fig. 5), this indication appears to be consistent with the idea that the amino acid residues 10 to 25 (containing all the arginine and 2 of the 3 lysine residues) immobilize upon binding of (Ap),A. This idea is supported by the fact that other proton resonances of the first seven amino acid residues appear as sharp peaks in the spectrum in Figure 2 trace D, i.e. Val CYH (-l.Op.p.m.), Thr CYH (~1.2p.p.m.), Lys CYH (N 1.4 p.p.m.), acetyl group of Serl (-2.1 p.p.m.). The high-field shoulder of the Val CYH resonance at - 1.0 p.p.m. is identified as Leu C6H (Vriend et al., 1985). However, this signal is not sufficiently resolved to decide whether Leu8 remains mobile upon addition of (Ap)sA, or not. Therefore, no definite conclusions can be drawn about the state of the amino acid residues Leu8 and Thr9. Figure 3 shows the result of the addition of double-stranded (A-T), to empty protein capsids at pH 5.0. Although many protein peaks in the 1 to 5 p.p.m. region overlap with (A-T), peaks, it can be seen that the same amino acid residues remain mobile as is observed for the titration with (Ap)sA. In this case also, approximately one molecule of (A-T), binds to one N-terminal arm (based on monomers). As a control experiment, addition of (Ap),A and (A-T), to protein lacking the N-terminal arm, in the form of empty capsids at pH 5.0, has been studied by n.m.r. spectroscopy. From the fact that no effect on the n.m.r. signal of the oligonucleotides is observed, it is concluded that these oligonucleotides bind only to the N-terminal arm. From the n.m.r. experiments, it is clear that the N-terminal arm of CCMV protein is necessary for oligonucleotide binding. The fact that the oligonucleotides (Ap),A and (A-T), can bind to the
m
Sheet
/vv\
Turn
JJj,
Figure 6. Secondary structure predictions for the E-terminal arm of CCMV protein. C+F, the method of Chou & Fasman (1974aJ). B et al., the method of Burgess et al. (1974). LIM, the method of Lim (1974uJ). C+F (mod.), the method of Chou & Fasman (1974aJ) after lowering the a-helix potentials for the arginine and lysine side-chains by 25%. Predictions C+F and B et al. simulabe neutral arginine and lysine side-chains. Predictions C+F (mod.) and LIM simulate charged sidechains.
empty capsids suggests that the N-terminal arms are accessible from the outside of the capsid. This is in agreement with binding studies of CCMV RNA to empty capsids (Bancroft, 1970). No significant, differences in binding characteristics (stoichiometry, immobilization, amino acids involved) are found for (Ap),A and (A-T),. This indicates that in vitro there is no specificity for deoxyor ribonucleotides or for the base sequences. This is in good agreement with other results (Bancroft et al.. 1969). (b) Secondary
structure productions
Figure 6 shows the result of the secondary structure prediction for the N-terminal arm of the coat protein of CCMV, using the method of Chou & Fasman (1974aJ) and the result using the method of Burgess et aE. (1974). It is seen that an a-helix is predicted from approximately residue 10 to 20. This prediction is in good agreement with that for the coat protein of another bromovirus, brome mosaic virus (Argos, 1981). This is not surprising, since the primary structure of the N-terminal arm of this virus protein and that of CCMV protein are quite similar. Also the N-terminal arm of the coat protein of brome mosaic virus has been found to immobilize upon binding RNA (Vriend et al., 1982). In Figure 7, a double a-helical net representation (Lim? 1978) of the N-terminal arm is given. This shows that an a-helical fold would bring all positive charges in close spatial proximity, which is very unlikely to occur. The fact that an a-helix is nevertheless predicted is readily explained from the nature of these two prediction methods. Both calculate from a small set of known structures the probabilities that amino acids are found in helix, sheet, turn or random-coil conformation. The probabilities, or structure potentials, are then applied to the amino acid sequence for which the secondary structure has to be predicted. The datasets used to derive the structure potentials,
Role of the N-terminal Arm
in CCM V Assembly
457
(c) Energy N
N
R
T
R
A L
G
' S
T
v
G
' S
v T
Figure 7. A double cc-helicalnet representation (Lim, 1978)of the N-terminal arm of CCMV. In this net, 2 arms are placed next to each other, to enablethe observation of the a-helical conformation from all directions. The distancebetweenadjacent residuesis proportional to the distancebetweentheir C” atoms in an a-helix. One region containing all arginine (R) and lysine (K) amino acid residuesis hatched.
however, contain almost no clusters of charged residues. Actually, many arginine and lysine residues in these datasets are in a neutralized state. Therefore, these predictions correspond to the case in which the positive charges in the N-terminal arm are neutralized, as in the native virus. To simulate the effect of repulsive forces between the positive charges in the N-terminal arm, the a-helix potentials for arginine and lysine in the Chou & Fasman method (Chou & Fasman, 1974a) were lowered by 25%. The corresponding prediction is shown in Figure 6, as well as the result of the prediction using the method of Lim (1974a,b). The latter method is not based on statistics, but employs general principles of protein folding and stability. It explicitly treats arginine and lysine side-chains as large hydrophilic, for which quite a number of u-helix prohibition rules apply. These predictions clearly demonstrate a random-coil instead of the a-helix for the case in which arginine and lysine side-chains are charged, i.e. for the IV-terminal arm of the coat protein of CCMV free in solution. Argos et al. (1978) have shown that the a-helix parameters for arginine and lysine can change by approximately 20% by changing the database. This means that a decrease of 25% as taken in the modified Chou & Fasman method (C+F (mod.) in Fig. 6) is well possible. It is also found that the probability for the formation of an u-helix in the region from residue 10 to 20 rapidly diminishes on decreasing the a-helix potentials of arginine and lysine, leaving definitely no a-helix left at a decrease of 25%.
calculations
The secondary structure predictions indicate that the charges on the arginine and lysine side-chains determine the conformation of the N-terminal protein arm. To check this, energy calculations were performed as a function of these charges. Because the computational task required to perform even a limited grid search over the conformational space of the 25residue long N-terminal arm is far too large for the current generation of computers, we could calculate only the relative energy contents of a small number of structures. From laser Raman studies, it is known that some secondary structure is present in the N-terminal protein arm in the native virus (Verduin et al., 1984). Therefore, it was decided to take two starting conformations in the a-helix region, and three in the b-sheet region. In these starting conformations, all amino acid residues had the same dihedral angles. The starting values for these dihedral angles are indicated by a cross in the Ramachandran plots given in Figure 8. From these starting conformations, energy minimizations proceeded for three different values of the partial charge p on the arginine and lysine side-chains. The dihedral angles of the residues in the structures obtained after the energy minimization are indicated in these Ramachandran plots. Table 1 shows the relative energy contents for these final structures. It can be seen that the u-helix conformation is energetically more preferable than the P-sheet when the arginine and lysine side-chains are neutralized (p = 0), simulating the situation in the virus, where these side-chains have a strong electrostatic interaction with phosphate groups of the RNA. In the absence of RNA, arginine and lysine side-chains in the N-terminal protein arm are not neutralized, or only partly by counter ions in the solutions. These two situations are simulated by p = 1.0 and 0.5, respectively. Although the a-helix remains the favourite structure with fully charged arginine and lysine side-chains, it is clearly seen that t’he energy differences between the several structures become less significant with increasing charges. This implies Table 1 Minimized energy content of some u-helix and p-sheet conformations of the N-terminal part of CCM V protein as a function of the partial charge (p) on the side-chains of the arginine and lysine-residues
Startingvalues Conformation LX1 a11
1 cal=4.184J.
Energycontent(kcal/molt) relative
~~
to an arbitrary reference point -~~~~~~~~ p = 0.5 p=
4
i
~~~~ p=O
-57 - 50
-57 -50
-227 -238
-126 - 127
63 51
-188 -169 -155
-71 -80 -71
87 66 67
-30 -50 -150
;:I /?I11 t
of the dihedral angles (deg.) ~-
150 150 150
1.0
c
”
a1 (I.01
a1 (01
a1 (0.5)
k$4=(-52-57)
b$,W=(-57,-57)
@,qJ)=(-52-57)
E=-227
E=-126
E=63
i 0 -
e
0
aII(O)
aII(O.5)
aII(1.0)
MqJI=(-50,-50)
(+,+1=(-50.-50)
(~,+)=(-50,-50)
E=-127
E=51
P
I
I
E=-238
T
c PI(O.5)
DI (I.01
(4?44=(-30.150)
k$,$JH-30350)
E=-71
E=87
p1r (01 NyJJ)=~-5o,l50) E=-169
I
0
pII1 (1.0) ~4!4J)=(-l5o,l50) V
E=67 -180
0
180 9---
Role of the N-terminal
Arm
CCMV
Assembly
459
has been suggested that double-stranded nucleotide helices are required for binding to the N-terminal arm of CCMV protein (Hemminga et al., 1985).
that the N-terminal arm is mostly in a random-coil conformation in solution, in accordance with the secondary structure predictions. This observation does not result from the fact that different local minima were obtained in the presence or absence of charges. This was checked by toggling p between 0 and 1.0 after completing the minimization and of the minimization subsequent continuation procedure. The values for the energy content obtained in this way were only slightly larger (i.e. up to +3I kcal/mol for fir with p changed from 1.0 to 0 (1 cal = 4.184 J)) than the values given in Table 1. From Figure 8 it can be seen that, after energy minimization, the residues 1 to 7 often have dihedral angles strongly deviating from the rest of the peptide chain. This probably arises from the presence of glycine residues at positions 4 and 6. It is known that glycine can cause structural irregularities (Lim, 1974a,b). From the calculations it is found that 75% of the differences in energy content in Table 1 is determined by electrostatic interactions, the remaining 25% arising from non-bonded and torsion interactions. This means that the electrostatic terms in the calculations are the dominating factors for the conformational changes, justifying our approach.
4. General
in
The authors are indebted to ProfessorJ. H. van Boom for providing (A-T),. We gratefully acknowledge the technical assistance of Miss H. Bloksma with the virus preparation. The authors thank the staff of the Computer Center for their continuous support during the many hundreds of hours CPU time needed for this work, and Miss M. Gerritsen for her assistance with the installation of the UNICEPP program package and the energy minimization calculations. We enjoyed stimulating discussions with Professor T. J. Schaafsma. This research was supported by the Netherlands Foundation for Biophysics, with financial aid from the Netherlands Organization for the Advancement of Pure Research (ZWO). The 500MHz ‘H n.m.r. investigations were supported by the Netherlands Foundation for Chemical Research (SON) with financial aid from ZWO.
References Abad-Zapatero, C., Abdel-Meguid, S. S., Johnson, J. E., Leslie, A. G. W., Rayment, I.. Rossman, M. G., Suck, D. & Tsukihara, T. (1980). &&re (London), 286, 33-39. Argos, P. (1981). Virology, 110, 55-62. Argos, P., Hanei, M. & Garavito, R. M. (1978). FEBS Letters, 93, 19-24. Bancroft, J. B. (1970). Advan. Virus Res. 16. 99-134. Bancroft, J. B. & Hiebert, E. (1967). VimoZogy, 32, 354356. Bancroft, J. B., Hills, G. J. & Markham, R. (1967). Virology, 31, 354-379. Bancroft, J. B.. Wagner, G. W. & Bracker. C. E. (1968). Virology, 36, 146-149. Bancroft, J. B., Hiebert, E. & Bracker, (‘. E. (1969). Virology, 39, 924-930. Burgess, A. W., Ponnuswamy, P. K. & Scheraga, H. A. (1974). Isr. J. Chem. 12. 239-286. Chidlow, J. & Tremaine, J. H. (1971). J’irology, 43, 267278. Chou, P. Y. & Fasman, G. D. (1974a). Biochemistry, 13, 211-221. Chou. P. Y. & Fasman, G. D. (19746). Biochemistry, 13, 222-245. Dasgupta, R. C Kaesberg, P. (1982). ;Vucl. Acids Res. 10, 703-713. Dunfield, N., Burgess, A. W. & Scheraga. H. A. (1975). J. Phys. Chem. 82, 2609-2616. Finch, J. T. & Bancroft, J. B. (1968). Nature (London), 220, 815-816. Harrison, S. C., Olson, A. J.. Schutt, C. E., Winker, K. K. & Bricogne, G. (1978). Nature (London), 276, 36% 373. Hemminga, M. A., Datema, K. P.. Ten Kortenaar, P. W. B.. Kriise, J., Vriend, G.. Verduin, B. J. M. & Koole, P. (1985). In Magnetic Re,sonance in Biology and Medicine (Govil, G., Khetrapal. C. L. & Saran, A., eds), pp. 53-76, Tata McGraw-Hill. New Delhi.
Discussion
Binding of (Ap)sA or (A-T), to empty protein capsids results in immobilization of the amino acid residues 10 to 25 in the N-terminal arm. Secondary st,ructure predictions and energy calculations have indicated that, upon neutralizing the positively charged arginine and lysine side-chains of the N-terminal arm, an a-helix is formed between residues 10 and 20. This corresponds well to the region that is immobilized, suggesting that an m-helix is formed upon binding the oligonucleotides. Similar a-helix conformation may occur in native virus and, if so, the suggested snatch-pull interaction model may be a representation of the assembly model in viva (seeFig. 1). The fact that in the predictions the amino acid residues 21 to 25 of the N-terminal arm in empty protein capsids do not form an a-helix but immobilize in the n.m.r. experiments, may be explained by noting that the N-terminal arm is fixed to protein “bodies” assembled in a capsid that performs a slow rotational motion. In the a-helix, all arginine and lysine side-chains are located at one side, facilitating binding to a regular array of negat
Figure 8. Ramachandran plots of the u-helix (aI( aII(p)) and b-sheet (PI(p), pII( pIII(p)) different values of the partial charge p. X, Starting point of the conformations with dihedral angles angles after energy minimization. Amino acid residues whose dihedral angles deviate significantly values are indicated by name. In most cases, these residues are in the region from residue 1 to 7. The rontent E is in kcal/mol relative to an arbitrary reference point (see Table 1).
conformations at (4, II/). 0, Dihedral from the starting minimized energy
G. Vriend et al. Hilbers,
C. W.
(1979).
In
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by M. F. Moody