Eur. J. Biochem. 93, 333 - 337 (1979)

Binding of Oligonucleotides to the Disk of Tobacco-Mosaic-Virus Protein James GRAHAM and P. Jonathan G. BUTLER

Medical Research Council Laboratory of Molecular Biology, Cambridge

(Received August 29, 1978)

The trinucleoside diphosphate A-A-G and the hexanucleotide fraction from a ribonuclease I digest of yeast RNA have been soaked into crystals of the disk aggregate of tobacco mosaic virus protein. At high concentrations these cause disruption of the crystal, probably by mimicking the normal nucleation of assembly.

At lower nucleotide concentrations the crystals remain intact and the differences caused by nucleotide binding have been studied by X-ray diffraction. The most obvious change is an upwards movement of about 0.3 nm at the low-radius end of the left radial helix in the protein with some stiffening of the helix so that it now extends visibly in from 4 nm to 3 nm radius. Similar shifts also occur in the right radial and left slewed helices.

A positive peak, which is tentatively identified with the bound oligonucleotide, is seen around 4 nm radius and below the right radial helix. The amino acid residues in possible contact with this feature are discussed.

The protein disk of tobacco mosaic virus (TMV) is an aggregate of considerable importance because of its role in the assembly of the virus particle [l]. As- sembly is initiated when the disk interacts with a unique sequence of bases internally on the RNA [ 2 , 3 ] . Crystallographic investpations have now revealed the structure of most of the subunit at atomic resolution [4,5]. However, the structure of that part of the sub- unit which lies at low radius in the disk is unknown because the protein in that region is not crystallo- graphically ordered [4] and is probably in constant motion [6 ] . The boundary between the ordered and disordered protein is at a radius of about 4 nm, which is the known radius of the RNA in the virus [7]. This ‘floppy’ region of protein may facilitate the incorpora- tion of the RNA which, once bound, may in turn tend to stabilize the protein.

This report describes the use of crystallographic techniques to try to investigate the mechanism of RNA binding to the disk. Small oligonucleotides were

Abbreviations. TMV, tobacco mosaic virus; CGMMV, cu- cumber green mottle mosaic virii\* A-A-G, adenylyladenylylguano- sine; A-A, adenylyladenosine.

Enzymes. Polynucleotide phosphorylase (EC 2.7.7.8); ribo- nuclease TI (EC 3.1.4.8); alkaline phosphatase (EC 3.1.3.1); ribo- nuclease I (EC 3.1.4.22); polynucleotide 5’-hydroxyl-kinase (EC 2.7.1.78); nuclease PI (EC 3.1.4.-)

diffused into the crystals and the structure investigated by the difference Fourier technique, as used for sub- strate binding to a crystalline enzyme. In this case, however, the exact ‘substrate’ is ill defined. The region of the RNA coated very early in assembly (the nucleation region) [2] has been sequenced [8], but shows no single unique feature likely to bind with a high degree of specificity to the disk. However, three nucleotides of the RNA are associated with each protein subunit, so that a repeating triplet of nucleo- tides might be expected. While there is not an absolute repeat over a long distance, a triplet motif, with guanosine (G) in every third position and usually adenosine (A), but sometimes uridine (U), in the other position is observed in the minimum core [2,8]. More- over, rebinding of partial nuclease digestion products of the nucleation region to the disk in solution showed strong binding only of fragments longer than about five nucleotides, although with little evidence of sequence specificity (H. Guilley and D. Zimmern, personal communication).

For the crystallographic investigations we there- fore tested two oligonucleotides ; the specific tri- nucleoside diphosphate, A-A-G, and a hexanucleotide mixture (also with the terminal phosphate removed) from a ribonuclease I digestion of yeast RNA.

334 Nucleotide Binding to TMV Protein Disk

MATERIALS AND METHODS

Oligonucleotide Preparation

A-A-G was prepared from A-A by reaction with a 3-fold excess of GDP in the presence of poly- nucleotide phosphorylase (PL-Biochemicals) and ribo- nuclease TI in 0.2 M glycine buffer, pH 9.3, 10 mM magnesium acetate, 0.1 mM CuSO4, 0.4 M NaCl for 30 min at 37 "C [9]. The reaction was stopped by heating at 100 "C for 15 min, the residue redissolved in water and treated with alkaline phosphatase to remove the terminal phosphate. The product was purified by binding to DEAE-cellulose in Tris chloride, pH 8.0, ionic strength 0.1 M, washing with water to remove unbound nucleoside, and elution with 30% (v/v) triethylamine brought to pH 8 with COZ. After repeated drying, the A-A-G was desalted over Sepha- dex G-25 into 0.1 M acetic acid and freeze-dried. The trinucleotide was characterised by labelling a sample with [Y-~'P]ATP (Radiochemical Centre, Amersham, Bucks.) and polynucleotide kinase. It was found to comigrate with authentic A-A-G and was further checked by partial digestion with nuclease PI and two-dimensional homochromatography [lo], confirm- ing its sequence as A-A-G.

The hexanucleotide fraction from an alkaline phosphatase-treated ribonuclease I digest of yeast RNA was isolated by chromatography on DEAE- cellulose in 0.02 M Tris chloride, pH 8, 7 M urea with a sodium chloride gradient from 0.05 M to 0.6 M. The various peaks were pooled and samples taken and labelled with [Y-~'P]ATP and polynucleotide kinase. They were then characterised by homochro- matography and found to contain essentially discrete length nucleotides in each pool, over the range from mononucleotide to octanucleotide. The hexanucleo- tide peak was treated in the same way as the product of the A-A-G preparation, and will contain random mixtures of five purine residues (A or G) with a single pyrimidine residue at the 3'-hydroxyl terminus.

Preparation of Derivatives

Crystals were soaked in the A-A-G and hexa- nucleotide at concentrations of 1 mM and 2 mM respectively. Raising the concentration of the A-A-G solution above 1 mM resulted in a dissolution of the crystals. The nucleotides were added in the Tris nitrate, pH 8.0, ionic strength 0.1 M, 0.3 M am- monium sulphate buffer from which the crystals had been grown, and so the addition produced no significant change in pH or ionic strength.

X-Ray Data Collection

Diffraction data to 0.5-nm resolution were collected from these crystals by rotation photography [I 11 using Franks-type X-ray focusing optics [12]. No significant

changes in unit cell dimensions were observed after soaking, but only variations within the range normally seen between films within the same data set. The R factors' of these data sets were 0.088 and 0.066 for the hexanucleotide and A-A-G data sets respectively. Full details are given in [13].

Calculation of Difference Maps

The native data set used for the calculation of difference Fouriers was extracted from the 0.28-nm data set of Bloomer et al. [ 5 ] . The mean relative difference in amplitudes ( A F / F ) of the derivative data set compared with native was 0.15 for the hexa- nucleotide data and 0.08 for the A-A-G data. The non-crystallographic symmetry of the disk gives rise to redundancies in the data which may be exploited to generate phase information [14]. This approach was used to calculate the phases for the 0.28-nm map of the subunit [5]. In the case of a derivative structure the same technique can be used to calculate phases corresponding to the derivative amplitudes using the native phases as a starting point. It is then possible to calculate a 'vector difference Fourier' whose coef- ficients are the true vector differences between the native and derivative structure factors, rather than the scalar differences in amplitude with the native phases as is normally the case in a 'difference Fourier'. The native and both derivative data sets were sub- jected to five cycles of phase refinement. (The native data set had been extracted from a higher resolution set and the estimated phases for this may not be quite appropriate, because they will be affected by the less reliable terms from the higher angle data.) In all cases the phase shift on the final cycle was about 1.75'. The final root-mean-square phase difference between the native and derivative structure factors was 9.3" for the hexanucleotide data and 6.0' for the A-A-G data. Vector differences were then calculated and each weighted by 1/(0; + 0;) for calculation ofthedifference map. (OD and q N are the standard deviations of the individual derivative and native amplitudes respec- tively.) An unweighted vector difference map was produced directly by simply subtracting the map of the native from that of the derivative after scaling the two on the regions of positive density.

RESULTS AND DISCUSSION

Effect of Hexanucleotide on Protein Conformation

Both the weighted and unweighted vector differ- ence maps showed essentially the same features but with lower weight in the unweighted map than the

' R factor = (,Z IF, ~ f i / N ) / / ( n , where the averages are taken over all reflections in the data set and where F is the mean of all the N symmetry-related reflexions F,.

N

z=1

J. Graham and P. J. G. Butler 335

Fig. 1 . Section of the electron density map, containing the disk axis and radius, showing the native Fourier in black and the hexanucleotide difference Fourier in white, dotted contours corresponding to negative dqjerence density. The grid lines are 1 nm apart. The native and difference densities are plotted on different scales; the native map is contoured at equal intervals of approximately 170 e/nm3 above the mean density, while for clarity the difference map is contoured at equal intervals of approximately 85 e/nm3. The left radial rod is marked LR. The difference map clearly shows that it moves upwards by about 0.3 nm, particularly at its low radius end and is extended by some 1 nm. The features above the left radial rod which also seem to move upwards are parts of the left slewed rod and hairpin bend (see text and Fig. 3)

Fig.2. A vertical section containing the radius passing through the right radial helix (marked RR). This can be seen to move upward,s. Also visible is the positive peak (marked n) without any corresponding negative peak, which we tentatively identify as the bound nucleotide

weighted one. The principal features of the hexa- nucleotide difference map are shown in Fig. 1 - 3, which are ‘vertical’ sections (i.e. parallel to the disk axis) through the difference map superimposed on the same sections of the native map. The rod-like positive feature in Fig. 1, together with its accompanying negative, are associated with the left radial helix (for terminology, see [4]) and indicate clearly that the oligonucleotide causes the cc-helix to move upwards by about 0.3 nm particularly at low radius, where it can be seen that the shifted protein is also ‘stiffened’

somewhat so that the chain now extends inwards to a radius of about 3 nm rather than fading by about 4 nm as in the native disk. A similar upward shift is associated with the right radial helix (Fig. 2). The left slewed helix also tends to move upwards particularly at its inner end (Fig. 3). This is not surprising, since the hairpin bend connecting the slewed rods lies directly above the low radius end of the left radial helix (see [ S ] ) .

The A-A-G difference map shows very similar features, particularly that associated with the left

336 Nucleotide Binding to TMV Protein Disk

Fig. 3. A skew-section, parallel to the disk axis containing rhe left slewed helix (marked LS) . This also tends to rise

radial rod, but the signal-to-noise ratio is much lower in the case of the A-A-G map (shown by the smaller value of A F / F ) rendering interpretation less straight- forward. The features of the vector difference maps discussed are those seen with both derivatives.

Possible Site of Nucleotide Sinding

The protein shifts clearly show that binding of the oligonucleotide is occurring and so we looked for a positive peak, without an accompanying negative, which might be the nucleotide. One complication is that the occupancy of the bound nucleotide may well be lower than the apparent weight of the protein shifts, since co-operativity between subunits is likely, so that binding to one subunit may affect several subunits in the ring. However, despite this, a discrete positive peak, with a height of two contours, is visible below the right radial helix (Fig. 2), again clearer on the hexanucleotide than on the A-A-G map. Exami- nation of the complete map does not show a corre- sponding negative peak and its position is close to that expected for the RNA in the complete virus [7]. We therefore suggest that this is probably the bound nucleotide, although it is not, of course, certain that even if this identification is correct, the whole nucleo- tide is visible. The crystals are grown from ammonium sulphate solution and a site where phosphate groups bind in vivo would probably bind a sulphate ion in the crystal. On oligonucleotide binding, the displace- ment of a sulphate by a phosphate would produce little difference in density, probably below noise level. Moreover, if any part of the bases or ribose residues were not rigidly fixed, they would not be visible either.

On the basis of this tentative identification, we have looked at the amino acid residues which would be in contact with the nucleotide, based on the atomic model [5]. The closest contact is with threonine-89 in the right radial helix, and there is also contact with

glycine-85 and alanine-86 in the next turn of the helix. The glycine residue is fully conserved in all TMV strains for which the sequence is known, while the threonine only changes to serine in one distantly related strain (cucumber green mottle mosaic virus, CGMMV). The alanine residue is less conserved, but again the mutations are only conservative, to serine or threonine. The other close contact is with the left radial helix of the neighbouring subunit. This is largely with valine-119, which is almost fully conserved (con- servatively mutated to threonine in CGMMV), and also with the methylene groups of the side-chain of arginine-122 and with serine-123. The arginine residue is again largely conserved, only changing to histidine in CGMMV and the cowpea strain of TMV. There is a more distant contact with the other side of the left radial helix of the same subunit to whose right radial helix the nucleotide is bound. This is with a highly conserved region, consisting of alanine-117, alanine-120 and isoleucine-121 (changed to alanine in CGMMV). Although this contact is rather long in the disk crystal, the change in tilt of the subunits on transformation from the disk to the virus helix [4] would tend to bring the left radial helices of adjacent subunits closer together in this region and so might improve the interaction. It is very noticeable that, at least in the closely related strains of TMV, seven of the nine amino acid residues in contact with this putative nucleotide are totally conserved. Further confirmation of the identification and elucidation of details of the contacts must await progress to higher resolution than the present 0.5 nm.

Consequences for Virus Assembly

All the main features in the difference map are in the A ring of the disk (i.e. that nearer the crystallo- graphic diad axis) and are mainly concentrated in the lower radius region of the axial rods. The region of

J . Graham and P. J. G. Butler 331

the map corresponding to the B ring has no significant features. While this could be an artefact of the crystal packing with interactions across the plane of the diad enhancing effects in the A ring, the disk is asymmetric with respect to the two rings and asymmetry of inter- action with the RNA is to be expected. RNA binding may well occur initially to one ring only with binding to the other only after ‘dislocation’. From our results, the initial binding is on the underside of the A ring, possibly in the neighbourhood of the right radial rod at 3.5 - 5 nm radius.

During virus assembly, binding of the RNA to the disk causes a major quarternary structure change, ‘dislocation’, from the two-layer disk to a two-turn helix. The changes observed here may well be the initial stages of this dislocation, the complete change being prevented by the lattice forces. The protein readily undergoes changes of this type : a lifting of the left radial helix is also observed on increasing the ionic strength of the ammonium sulphate solution [15].

The greater change caused by the random purine- rich hexanucleotide mixture, than by the possibly specific trinucleotide, A-A-G, is slightly puzzling. Possibly a minimum length of nucleotide (longer than three) is required to produce major changes. The hexanucleotide mixture used may be rather non-spe- cific in its binding making it crystallographically indistinct. Further work is in hand using the specific hexanucleotide, A-A-G-A-A-G.

During the course of most of this work, J .G. was supported by a Medical Research Council scholarship for training in research methods. We thank Christine Harley and Claudio Villa for technical assistance and Hubert Guilley and David Zimmern for telling us of their unpublished results.

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J. Graham, Department of Medical Biophysics, Victoria University of Manchester Medical School, Stopford Building, Oxford Road, Manchester, Great Britain, MI 3 9PT

P. J. G. Butler*, M.R.C. Laboratory of Molecular Biology, Postgraduate Medical School, University of Cambridge, Hills Road, Cambridge, Great Britain, CB2 2QH

* To whom correspondence should be addressed.