Location of Tyrosine Residues in the Disk of Tobacco-Mosaic-Virus Protein and Comparison of the Subunit Packing with that of the Virus

Eur. J . Biochem. 83, 523-528 (1978)

Location of Tyrosine Residues in the Disk of Tobacco-Mosaic-Virus Protein and Comparison of the Subunit Packing with that of the Virus

James GRAHAM and P Jonathan G BUTLER

Medical Re5earch Council Laboratory of Molecular Biology, Cambridge

(Received August 23, 1977)

The products of iodination of the disk of tobacco mosaic virus coat protein have been investigated in order to locate the reactive amino acid residues in the three-dimensional structure. Reaction occurs mainly with tyrosine-I39 and, to a lesser extent, tyrosine-:! and the positions of these modified residues have been determined by X-ray crystallography. Different extents of reaction are found in the two rings of the disk and also, on adding the high salt concentration needed for stabilisation of the crystal during reaction, some conformational changes in the polypeptide chain in the inner part of the disk.

Comparison of the relative positions of residues 27 and 139 in the disk and virus shows that little distortion occurs in the outer part of the subunit during the transition between the disk and virus structures.

Tobacco mosaic virus (TMV) was the earliest known biological system capable of self-assembly. This capacity confers a special interest on the investi- gation of the structure of the particle. The intact virus consists of a helical rod of coat protein subunits, having 3 6'/3 subunits per turn, with the single-stranded RNA intercalated between successive turns at a radius of 4.0 nm (for a review see [l]). This structure is ex- tremely compact, with the protein subunits in tight contact, affording maximum protection to the RNA. It has been determined to a resolution of about 0.7 nm by X-ray diffraction from oriented gels [2].

The protein aggregation is highly polymorphic, leading to many other aggregates besides the complete virus, the most biologically important of these being the disk. This consists of two rings, each of 17 subunits, stacked in a polar fashion [3]. It is essential for initia- tion of the nucleoprotein helix [4] and can act as the protein source for its elongation (for a review see [ 5 ] ) . Crystals of TMV protein suitable for X-ray diffraction, with the disk as the asymmetric unit, were obtained by Leberman et al. [6] and have been investigated by X-ray crystallography to a resolution of 0.5 nm by Champness et al. [7]. These authors have proposed a tentative chain tracing in which the subunit structure at intermediate radius is composed of four roughly parallel a-helical rods, two lying approximately radially and two slewed with respect to the radial direction.

Ahhrevintion. TMV, tobacco mosaic virus

In the interpretation of such maps, it is useful to know the positions of some defined residues, and these can be determined by labelling with heavy atoms. Several such positions are known in the virus [2], but so far only one in the disk, that of cysteine-27 [3]. The determination of other positions would enable comparisons of vectors between residues in the disk and virus which would indicate whether or not there is any subunit distortion in the transition between the two aggregates. The tyrosines provide possible sites for such labelling. There are four tyrosine residues (2 , 70, 72, 139) in the polypeptide chain of common strain TMV and it has been shown that in the intact virus tyrosine-1 39 reacts readily with iodine, while in the dissociated protein all four tyrosines react at varying rates (139 > 2 =. 70, 72) [8]. Cysteine-27 is the only other residue to react with iodine. This paper describes the location of two of these tyrosine residues in the protein disk by X-ray analysis of iodine-labelled crystals.

MATERIALS AND METHODS

lodination of Crystals Crystals, prepared as previously described [6],

were soaked in a solution of 5 mM IZ with 0.1 M KT in the crystallising solution. In order to prevent the crystals dissolving in the iodine solution, it was necessary to increase the ammonium sulphate concentration of the mother liquor from 0.3-3 M. The crystals

524 Location of Tyrosines in the TMV Protein Disk

were left in this solution for times ranging from overnight to one week before being selected for X- ray analysis.

Crystallogr.riphj

The X-ray data were collected by precession photography in the form of three centrosymmetric projections, to a resolution of 0.65 nm. Two of these projections are on zone axes perpendicular to the 17- fold rotation axis of the disk. The non-crystallographic 17-fold rotational symmetry can be used to generate a three-dimensional difference Fourier map at low resolution (about 1.5 nm in azimuth and 0.8 nm in 2) using the method of Gilbert [9] which is similar to the methods used for reconstructing three-dimensional images from electron micrographs. The native structure factors used for the calculation of difference coefficients were taken from the 0.5-nm data set of Champness rr ul [7] .

Locution qf‘ Reactive Residues in Amino Acid Sequence

Crystals in six separate vials were reacted with iodine as described above for 24 h, but with 5 pCi 1251, added as sodium [‘251]iodide (Radiochemical Centre), per crystallisation vial (about 8 mg TMV protein). The reaction was terminated by the addition of excess sodium thiosulphate. The crystals were then washed clean of mother liquor and as much conta- minating precipitated protein as possible, before dissolving in 8 M urea, Tris . CI, ionic strength 0.1 M, pH 8.0. The protein was reacted with iodoacetate (2 mM) at 20 “C for 2 h and the reaction stopped by the addition of excess 2-mercaptoethanoI. Each sample of the protein was then dialysed against several changes of 1 mM hydrochloric acid.

Trypsin (0.1 mg) was added to the suspension of precipitated protein and the pH adjusted to about 8 by the addition of solid ammonium hydrogen car- bonate, before digestion for 4 h at 37 “C. The precipi- tates dissolved during the first 30 min. The resulting peptides were freeze-dried and then fractionated by electrophoresis on paper at pH 6.5 and the radioactive peptides located by autoradiography. These were then further purified by paper electrophoresis at pH 3.5 and thereafter characterised and identified by amino acid analysis, amino-terminal identification and, in one case. carboxypeptidase A digestion.

RESULTS

Locurion of Iorlinc .4 toms in the Crystal

Crystals responded in one of two ways to soaking in the iodine solutions: either they turned brown,

removing all the colour from the solution overnight, or they turned pale yellow overnight, both crystals and solution turning colourless in the course of a week. The brown crystals, although remaining well formed, did not diffract; the others diffracted well.

A difference Fourier map of the c-axis projection, calculated using data collected from a crystal soaked overnight in iodine, showed a ring of positive density at high radius (about 7.5 nm) (Fig. 1). When data were collected after a soak of several days these features no longer appeared. This suggested to us that iodine is a very labile ligand in these conditions, and that the X-ray data had to be collected within a day or so of soaking. In particular, it seemed essential that the a and b axis projections, which are used for the three- dimensional image reconstruction, should be collected simultaneously. Even when this precaution was taken, however, inconsistencies were observed between re- flexions common to both projections, indicating some variability from crystal to crystal within a single vial.

Because of these inconsistencies, X-ray data were collected from control crystals soaked at the high salt concentrations used in the iodination experiments, both with and without KI, in the absence of iodine. The average structure factor changes (dF;F> for the three centrosymmetric planes were 35 for hk0, 40 % for h01, 38 % for Okl in the case of the iodinated crystals. For the control crystals, these changes were 25 %, 27 ”/, and 32 % respectively.

An additional difficulty arose from the fact that the iodinated and control crystals were not strictly isomorphous with the native. Subject to some variability from crystal to crystal, the average unit cell dimensions from both types of crystals were a = 22.65 nm, b = 22.5 nm and c = 17.25 nm. (The native crystals have unit cell dimensions a = 22.8 nm, h = 22.4 nm, c = 17.4 nm.) Considering the low resolution of the reconstructed maps, these changes were not considered serious.

Despite the inconsistencies in the data, reconstructions computed from data collected in different experiments soon after soaking showed several reproducible features. Besides a number of positive peaks, attributable to iodine. there were also a number of regions of positive density with characteristic shapes associated with negative regions. We attribute these to small changes in the protein conformation. These features also appeared in difference Fouriers computed with the control data showing that iodine binding. is not necessary to produce them.

The features of the ‘protein movements’ which are most readily interpretable at this resolution are the following.

a) A long rod of density stretching radially (cp = - 11”) from R = 4.5 nm to R = 7.0 nm and tilting slightly downwards from Z = - 1.4 nm at the inside to Z = - 1.65 nm at the outside. This is accompanied

3. Graham and P. J. G. Butler 525

Fig. 1. Differcwce electron dtwsity map ( I 7-Jdd averuged) of the c-axis projection of an 12-labelled crystal. Dashed lines represent negative contours. The grid lines form 1.9-nm squares. The rings of density with 7.5-nm radius (labelled I) correspond to both iodine sites which are not resolved in this proiection. The density at intermediate radius arises from protein movement, particularly of the left slewed helix (see text . " and Fig. 3 )

by a similar negative feature 0.5 nm below it (cp is taken as the direction of the crystallographic diad axis, and 2 is measured from the plane of the diad, negative values are given in order that the polarity of the disk is consistent with that of the virus map, see [2].) We interpret this feature as an upward movement of the left radial rod (in the notation of Champness et al. [7], see Fig.2).

b) A 'hook-shaped' peak at Z = - 1.0 nm, cp = 0". This is shown in Fig. 3 A. The peak is elongated, stretching from R = 4.0 nm to R = 6.5 nm at an angle of about 10" to the radius vector, then turning through about 90" towards positive cp for another 1.5 nm. It is accompanied by a negative feature of similar shape which is slightly higher in 2 (2 = - 0.9 nm) and displaced in cp by about + 10" relative to the positive peak. Fig. 3 B shows the corresponding section of the native map showing part of the left slewed rod. We interpret the positive negative pair as a sideways (and slight downwards) movement of the left slewed rod by about 3" towards negative cp. This corresponds to the rod moving sideways through a distance which is less than its width producing a

positive-negative pair some 0.5 nm apart with no difference density between.

There are other smaller features which also seem to be attributable to some small changes of the protein or bound solvent, but at this resolution we can offer no interpretation of these. It is noteworthy that we observe similar features during the binding of oligo- nucleotides to the disk in the crystal (unpublished work).

In difference Fourier maps calculated using the control crystals as native, feature (a) above is totally absent while the weight of feature (b) is considerably reduced (indicating that iodine binding may have some influence on this movement). Common features on both types of difference Fourier map lead us to attribute two distinct peaks to bound iodine, and the positions of these are shown in Table 1. Also shown are the positions of the methyl mercury binding site in the disk (Cys-27) [3] and the positions suggested for residues 27 and 139 in the virus [2]. Owing to the 'noisy' nature of the reconstructed difference Fouriers and the rather curious relative occupancies in the A and B rings, no satisfactory refinement of the heavy

526 Location of Tyrosines in the TMV Protein Disk

I 0 3 .O 6.0 9.0 0

-2 5 4 'I

iv

V

Fig. 3. Rridicrl \CI /ioir\ / / ru iug / i rhc rrc~onsrructcd c l i f f ~ w n c ~ r maps cor onding /O the A ring o f ' h d k k . The first t w o Scctions are at cp- 1.5 in the iodine-miked cryrtals (i) and the high-ionic-strength control and show the bound iodine peak ( a ) at 7.4 nm radius (labelled I ) . The rcmaiiiinp sections ai-c at cp= - 13 in the iodinated crystals (iii), the control (iv) and the 0.5-nm resolution protein map ( \ ) (see [7]). .The leli radial hclix (;it the bottom of v) moves upward by about 0.5 nm in iii and iv. These maps are at very low resolution and the reconstruction process intrtduces ;I high 'noise' content, particularly at low radius. This results in direrences in the weights of certain features and in the contouring h e 1

atom positions could be obtained and the coordinates quoted refer to the measured positions of the heavy atom peaks on the difference Fouriers.

The iodine peak of 7.4-nm radius (a) appears in all reconstructions (with rapidly collected data) and has the curious property of appearing in only one ring of the disk, the A ring, i.e. that nearer the crystallographic diad. This may be due to the crystal packing: the iodine position is near the surface of the subunit, and B rings of neighbouring disks make close contact in this neighbourhood and so may prevent or hinder the iodination reaction (cf. Fig.2 of [lo]).

The other higher radius peak (b) occurs at com- parable positions in both rings, although it is much weaker in the A ring. The weight of this peak is rather variable. We take the variability in occupancy in this site and perhaps in other unidentified minor sites as the reason for the inconsistencies in the data.

Residuues Reacted

From each of the vials, the major radioactive tryptic peptide (a l ) was basic and sometimes accompanied by a weaker satelite band (a2) migrating slightly f. 'ister at pH 6.5. All of the samples also contained some radio-

J . Graham and P. J . G. Butler 521

Table 1. Position.s qf iodine hindinx sites in the disk along with labelled residues Azimuth ( a , ) is measured from the direction of the crystallographic diad. Z is measured from the plane of the diad, negative so that the polarities of the disk and the virus are the same. Also included are the methyl mercury nitrate positions in both virus and disk and the position of residue 139 in the virus, acysteine in strain Ni2068 labelled with dimercury acetic acid. Holmes et al. [2] take this position to be their zero of a, and 2

Labelling agent Residue R for q for Z for labelled ~~ ~ .~ - ~ ~ ~~ ~ ~ ~~~ ~ ~~ ~~ ~ ~~~~~ ~ ~~~~~

ring A ring B virus ring A ring B virus ring A ring B virus

nm degrees nm

Methyl mercury

Iodine (a)

nitrate Cys-21 5.92 5.89 5.67 - 4.1 -8.0 16 - 3.21 -3.88 1.17

(dimercury acetic 0

12.0 9.0 - --0.7 -3.3 -

- 0 - 1.95 acid for virus) Tyr-139 7.4 - 1.2 - 19.7 -

Iodine (b) Tyr-2 8.0 7.8 -

Table 2. Characterisation qf ‘2sI-labelled peptides Ratios are those which give the closest to integer values for most residues. Other amino acids, and values shown as a dash, were present in ratios below 0.05. For ratios far from an integer, the value from the sequence assigned is shown in brackets beside the number of residues assumed. From amino acid sequence of TMV protein [I 11, the following sequences were assigned: peptide a t la2: Gly-Thr-Gly-Ser- Tyr-Am-Arg (residues 135 - 141); peptide b : Ac-Ser-Tyr-Ser-Tle-Thr-Thr-Pro-Ser-Gln-Phe (residues 1 - 10)

~ ~~ ~

Amino acid Peptide a l Peptide a2 Peptide b ~ -~ ~ ~~~ ~~ ~~~ ~

ratio assumed ratio assumed rdtio assumed residues residues residues

Aspartic acid Threonine Serine Glutamic acid Proline Glycine Alanine Isoleucine Leucine Tyrosine Phenylalanine Arginine

N-terminus ~~~~~ ~~

1.05 1.01 0.94 0.34

2.88 0.19

-

-

-

0.83

1 .OO -

~~

0.16 -

1.96 2 2.94 3 1.17 1 0.94 1

0.13 -

1.02 1 0.14 -

0.63 1 0.97 1

0.41 0-1 (0)

- -

~~ - ~~~ ~ ~~~

not found

active material which stayed at the origin at pH 6.5, which is the known behaviour of the tryptic peptides from residues 1 to 41 and from residues 123 to 134. This latter peptide does not, however, contain a tyrosine residue so that this is most probably labelling in peptide 1-41. From some of the digestions there was also a moderately acidic labelled peptide (b). On electrophoresis at pH 3.5, all of these peptides appeared to be pure, with peptides a1 and a2 migrating together, while peptide b was still slightly acidic even at pH 3.5.

The amino acid compositions and amino-terminal residues of the peptides are shown in Table 2. From these peptides a1 and a2 were identified as the tryptic peptide from residues 135 to 141, with the label probably attached to tyrosine-139, as is found in the intact virus [8]. The splitting of this band is probably due

to the formation of monoiodotyrosine and diiodo- tyrosine. The less reproducible minor peptide (b) corresponds best to a chymotryptic fragment from residues 1 to 10. Since this peptide would also have the acetylated amino terminus of the virus protein if it had been generated by cleavage at phenylalanine-10, it is compatible with the failure to detect any amino- terminal residue. This assignment was checked by carboxypeptidase A digestion, which liberated phenylalanine and glutamine, which is compatible with the label being on tyrosine-2. This identification is also consistent with the radioactivity seen at the origin in all of the samples at pH 6.5.

Iodine therefore seems to be reacting, under the conditions employed here with the crystals of the disk aggregate of TMV protein, mainly and reproducibly with tyrosine-1 39. To a lesser extent and more variably,

528 J . Graham and P. J. G. Butler: Location of Tyrosines in the TMV Protein Disk

it also appears to react with tyrosine-2. The identification of the major site with tyrosine-139 is consistent with the position found for this site in the virus mutant Ni2068 where Cys-139 was labelled with mercury [2]. We therefore identify these residues with the major and minor iodine sites (a and b) seen by X-ray crystallography as discussed above.

DISCUSSrO!v

One of the more curious features of the interaction of iodine with the disk is the non-reactivity of cysteine- 27. Fraenkel-Conrat [12] has observed that, in TMV, Cys-27 is readily oxidised to form a stable sulphenyl iodide group. The stability of this group is surprising since sulphenyl iodides are normally hydrolyzed to sulphonic acids. Fraenkel-Conrat attributed the stability in this case to the inaccessibility of the site to the aqueous soli,ent. In the disk, this site is certainly accessible to methyl mercurials (but not to larger phenyl mercurials). I t may be that in the disk aggregate the movement of the left radial rod during the conditions for the iodination reaction totally blocks this site.

Mandelkow and Holmes [13] have labelled the N-terminus of strain U2 with methyl mercury liganded with 4-sulpho-phenylisocyanate and give the coordinates of the heavy atom position as R = 8.85 nm. p = 7.0 , Z = 1 . I 2 nm. This may be compared with our proposcd position of Tyr-2, using Tyr-I39 as a reference point. The two positions are separated by about 1.0 nm which is not surprising considering the large ligand used in the virus.

A more striking relation between the sites in the disk and virus appears when the vector between sites 27 and 139 is compared in the two structures. Electron density maps of the disk and virus show that in the A ring of the disk the subunits are approximately perpendicular to the disk axis, while those of the B ring slope upwards towards a higher radius by about 10 I . In the virus, on the other hand the subunits slope downwards by about 10' (A. C. Bloomer, personal communication). As a result of this the ends of the virus particle are readily distinguishable in high- resolution electron micrographs, one end appearing convex while the other is flat or concave (cf. [14],

Fig. 2). During the transformation from disk to helix the orientation of the subunits of both rings must change involving at least a tilt of 10 and 20' for rings A and B respectively (discussed in [15]).

Examination of the data in Table 1 for the positions of residues 27 and 139 in the virus and ring A of the disk shows that in both cases their azimuthal separations are almost identical as are their radial separations. However, their separations in Z differ: 0.74 nm in the disk and 1.17 nm in the virus, corresponding to a downward tilt of the vector between residues 27 and 139 by 10.5'. These data suggest that the transformation of the A subunit from the disk to the virus in- volves a rigid body doNnward tilt with little distortion, at least between radii of 6.0 and 7.5 nm.

We thank the Medical Research Couiicil lix a Scholarship for Training in Research to J . G .

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J . Graham arid 1'. J . < i . Butler. M. R. C. Laboratory of Molecular Biology. Hills Road, Cambridge. Great Britain. C'B2 2QH

Documents

Location of Tyrosine Residues in the Disk of Tobacco-Mosaic-Virus Protein and Comparison of the Subunit Packing with that of the Virus