Protein-RNA interactions during the assembly of tobacco mosaic virus

4

TIBS -January 1980

Reviews Protein-RNA interactions during the assembly of tobacco mosaic virus K. C. Holmes There are still very few systems from which we can obtain detailed information about the geometry o f the interactions between proteins and nucleic acids. One such is Tobacco Mosaic Virus ( TMV). T M V consists of 214O protein subunits, each of mol. wt 17,420 (158 residues), arranged on a helix o f pitch 2.3 nm with 16 1/3 subunits per turn. Winding through this helix is a single strand o f RNA 6400 nucleotides long (Fig. I) [1,2].

Structural studies on Tobacco Mosaic Virus (TMV) have a long history. In 1936 Bernal obtained the first X-ray diffraction patterns from orientated gels of TMV. Using the theory of Cochran, Crick and Vand, J. D. Watson [31 was able to begin the analysis of the fibre diagram and showed that the structure of TMV was helical with 3n + 1 subunits every 6.9 nm. R. E. Franklin and D. L. D. Caspar took up the study of the structure of the orientated gels. Franklin was soon to be joined by A. Klug. After Franklin's untimely death in 1958 Klug took over the leadership of the group. In 1962 the virus group moved from London tothe MRC Laboratory for Molecular Biology in Cambridge where a great deal of the work described in the following text was carried out. Franklin started out with the ambitious resolve to determine the structure of TMV by means of the then newly discovered method of isomorphous replacement. It was quickly established that the virus had 49 subunits in three turns [4], that the virus was hollow, and that the nucleic acid was located at a radius of 4.0 nm [5]. These results were obtained in collaboration with Caspar and with Holmes by analysis of the low resolution part of the zero layer-line (< 1.0nm 1). Together with Klug, Franklin showed that the virus surface is marked by a deep helical groove. She proceeded to analyse the third layer-line in order to arrive at a low resolution helical projection of the virus. With the death of Rosalind Franklin in 1958 the project lost some impetus. Moreover, in 1958 the necessary methodology, particularly in computer development, was not available. As it turned out, determining the structure K. C. ltolmes is at the Afax-Planck.htstitut fiir medizinische Forschung, tteidelberg, F.R.G.

of TMV entailed developing new methods in theoretical and practical crystallography and in biochemistry so that nearly 20 years were to elapse before detailed structural information about the RNA and its environment could be obtained. Klug and Holmes continued the structural analysis of the orientated gels by means of the method of isomorphous replacement. In particular, in an unpublished study Klug was able to show that the RNA was single stranded at a time when it was widely supposed, on the basis of end-group analyses, that the RNA was in many segments. With the establishment of Klug's group in Cambridge the necessary technical milieu became available to extend Franklin's early work. TMV coat protein, which is capable of

Fig. 1. Diagram o f Tobacco Mosaic Vin~s drawn by D. L. D. Caspar [2]." the sLy.turn segment shown corresponds to 1/20 th o f the lengtt~o f the vinls. The RNA chain, with three bases per protein subunit, is coiled between the turns o f the 2.3 nm pitch helLr o f protein subunits. The diameter of dte RNA is 8.0 nm. There are 16 ! 13 subunits per turn. The inner diameter o f the particle is 4.0 nm and the outer diameter is 18.0 nm.

assembly in many polymorphic forms, reassembles to two-ring disks at neutral pH. Microcrystals of disks were first reported by Macleod et al. Reproducible crystals were produced by Finch et al. [6] in Cambridge. Furthermore, they were able to demonstrate that the symmetry of the disk was 17-fold. This work opened the way to obtaining an atomic model of TMV by means of X-ray crystallography. The disk consists of two rings of protein subunits, each with 17 subunits - the largest asymmetric unit yet solved. To solve the structure of the disk to 0.28 nm resolution it was necessary for Bloomer et al. [7] to measure the intensities of 2 x I{Y reflexions! The analysis showed the entire po!ypeptide chain except for a section within 4.0 nm radius which was not visible. This reflects the fact that in the disk (but not in the virus) there is a flexible segment of about 25 residues length. The electron density allowed all the residues to be fitted except for the flexible segment between 89 and 113 and the C-terminal residues 155-158. The preparation of heavy atom derivatives for the solution of the phase problem presented major difficulties so that much of the analysis was carried out with a single mercury derivative. Using the formulation of Crowther, Jack [8] was able to demonstrate the power of the non-crystallographic 17-fold symmetry as a supplement to the heavy atom method for the solution of the phase problem. By setting up the equivalent formulation in real space Bricogne [9] made it possible to use this method for the high resolution analysis of the structure of the disk [7]. Parallel to this work the analysis of the X-ray fibre diffraction progressed. In 1968 Holmes moved to Heidelberg and work on the intact virus was transferred there. The 9 Else~iertNorth-Itolland Biomed:i~l Press 1980

T1BS - J a n u a r y 1 9 8 0

Growth

determination of the structure of TMV by a strip of beta sheet. In the central refrom the X-ray fibre diagrams by means of gion of each subunit on the distal side of the the method of isomorphous replacement is beta sheet is a cluster of aromatic residues a unique problem which entails unscramblwhich gives rise to a continuous belt of hying cylindrically averaged data. The data at drophobic interactions encircling each ring each point in reciprocal space consist of the of the disk. At the proximal ends of the LR sum of squares of a number of Bessel funcand RR helices is a segment (89-113) tion terms, the actual number of Bessel which is highly mobile (the flexible loop). functions which contribute being determined by the helical symmetry of the partiThe structure of the virus cle ard the reciprocal space radius of the The structure of the subunit in the virus point in question. Theoretical studies by [13] (Fig. 4a) is substantially the same as Klug et al. [10] had shown that in principle the disk except for the flexible loop and the it is possible to regenerate the threevertical intersubunit contacts. Proximally dimensional structure if the problem of from 4.0 nm radius, where the density in 'separating the Bessel functions' can be the disk fades out, one finds the LR and solved. Franklin and Klug realized that, to RR helices continuing to a low radius a resolution of about 1.0 nm, on account of (2.5 nm) where they are joined together by the high symmetry of TMV (49-fold), only a strong vertical column of density (the one Bessel function could contribute to the V-column) which was identified by Stubbs intensity. Using this idea Barrett et al. [11] et aL [13] as an alpha helix although more were able to calculatea three-dimensional Fig. 2. Diagram of the encapsulation of RNA daring recent studies show this identification to be electron density map of the virus with a dregrowth of TMV partides [191 The dominant direcsomewhat uncertain. In addition to this nominal resolution of 1.0 nm. This map tion of growth (indicated by an arroyo is upwards density, which can unequivocally be showed the general appearance of the from the assembly origin (towards the 5' end}. The ascribed to the protein, there is a ribbon of nucleic acid running circumferentially at RNA is pulled through the hollow core of the nascent density running roughly circumferentially virus. 4.0 nm radius, and the overall appearance at a radius of 4.0 nm underneath the LR of the subunit including strong radial feahelix which can be ascribed to the nucleic tures which were later shown to be alpha- assembly origin) binds to the disk acid. A diagram of the RNA conformation helices. The structure of the virus extends (Zimmern and Butler [16], Zimmern [17], is shown in Fig. 4b. Each protein subunit proximally to a radius of 2.2 nm, in sharp Jonard et al. [18]) thereby mediating the binds three bases which are numbered 1,2 contrast to the situation in the disk. Later formation of the lockwasher and initiating and 3 from the 5' end. The triplet of bases Holmesetal. [12] analysed the data out to a the growth of the helix. The assembly form a claw-like structure round the LR resolution of 0.67 nm, which is the two- origin of the RNA is about 100 nucleotides helix. Two bases are at a radius of about Bessel-function limit. The resulting map long and the initial binding is thought to 3.8 nm and one base is at a larger radius showed some of the polypeptide chain, in occur between the turns of the disk. Close (4.5 nm). The phosphates are clustered particular the two radial alpha helices to the center of the assembly origin is the around a radius of 4.0 nm and appear to which are such a prominent feature of the sequence AGAAGAAGUUGUUGA bond to the adjoining RS and RR helices structure of the TMV subunit. Further UGA. In this and the surrounding sefrom the neighboring subunit in the next work admitting tl-,ree Bessel functions quences there is a strong tendency for every turn down. Thus the RNA binding site lies (Stubbs, Warren and Holmes [13]) yielded third residue to be G. The assembly origin between the rings of a disk, in agreement an electron density map at 0.4 nm resolu- has a very low C content and occurs about with the model for assembly proposed by tion by the use of six heavy atom deriva- 15% of the length away from the 3' end. Butleretal. [15]. tives, which taken together with the Growth takes place fastest in the 5' direcdetailed map of the subunit in the disk pro- tion. Rather unexpectedly, as growth produced by Bloomer et al. [7] yields a consid- ceeds towards the 5' hydroxyl end the 5' The RNA-binding site erable amount of stereochemical informa- end is dragged through the central hole of The best description of the RNAtion about the nature of the protein-RNA the nascent virus (Fig. 2) so that both the 3' binding site presently available is obtained interaction in TMV assembly. and 5' ends are initially at the same end of by combining the details of the disk structhe virus [19,20]. ture [7] with the 0.4 nm map of the virus Assembly of the virus [13]: an important component of the bindDuring the assembly of the virus the coat The structure of the disk ing site seems to be provided by the The general appearance of the subunit, invariant pair of aspartate residues protein has to specifically recognize the viral nucleic acid and at the same time has as deduced from the high resolution studies (115,116) on the LR helix. These probably to be able to tolerate the variation of of Bloomer et aL [7], is shown in Fig. 3. The hydrogen bond to the RNA ribose 2' OH sequence found when encapsulating the four main alpha helices, which run roughly on residues 1 and 3. Such a ribose binding 9RNA. Butler and Klug [14] showed by radially, account for about 60 of the 158 site is typical of alcohol dehydrogenase, kinetic analysis that the disk is the major residues. They are known as LR, RR, LS lactate dehydrogenase and malate dehyspecies responsible for coating the RNA and RS. The helices are packed as in a seg- drogenase; here this binding motif may be during in vitro assembly. Assembly starts ment of a left-hand~d, four-stranded used twice per protein subunit. The by the insertion of a specific RNA loop (the supercoiled bundle with hydrophobie con- accompanying bases find themselves in initiation loop) into the disk (Butler et al. tacts along the center. The distal ends of hydrophobic pockets close up against the [ 15]). A specific sequence of the RNA (the the four helices are connected transversely alpha helix, as was suggested by Stubbs et

I nucleatiOn

TIBS-January 1980

6

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al. [ 13]. Base 2 is at a larger radius and may form a hydrogen bond to Ser-123. The effect of the tight binding of RNA residues 1 and 3 to the LR helix is to pucker the RNA so that the phosphate residues are grouped together, thereby making them a target for the formation of salt bridges with arginine residues 90 and 92 from the RR helix of the subunit directly underneath. The assignment of the invariant residues 90 and 92 to the phosphate-binding site is plausible but higher resolution studies of the virus structure are needed before such an assignment can be proven. A hypothetical trigger mechanism for the disk-heli.x transition

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(B) Side view through a sector of the dL~ks/towing tlre disposition of the subunits in the A and B rings and tire axial contacts between them. There are three regions o f contact indicated by a solid line for the hydrophobic contact of Pro-54 with A/a-74 and Val-75, by dashed lines for the hydrogen bonds between Thr-5 9 and Ser-14 7 and-148 and fitnher dashed lines for the extended salt-bridge system. All these bonds are broken in tire transition to the helical smtcture of the virus. Tire proximal region of the subunit, which has no structure in tire disk form is shown schematically by a broken line in approximately the configuration it takes in tire virus [13].

The surprising result from the studies summarized above is that the major component of the RNA binding site in TMV is one alpha helix, the LR helix, which binds a triplet of bases. In the disk structure, which is the template for binding RNA, this helix continues as a finger proximally to a radius of about 4.0 nm (residue 114) before the electron density peters out. This finger could nucleate the binding of the RNA by means of three kinds of interaction: (a) A stereospecifie interaction of the aspartate groups 115,116 with two of the ribose groups; (b) A hydrophobic interaction with the three bases forcing them into the shape of a claw round the alpha helix (Fig. 4); (c) Specific hydrogen bonding with Ser-123 and other residues. The binding is probably accompanied by the formation of salt bridges between Arg-90 and -92 from the neighboring subunit one turn deeper in the virus helix and the phosphate groups of the nucleic acid. Initially 90 and 92 are part of the randomcoil segment adjoining the RR helix but apparently through the binding to the phosphate groups this structure is stabilized and as a result the V-column and the rest of the LR and RR helices which are not seen in the disk can build. However, the V-columns from adjoining subunits are in close contact and it seems probable that they can only fit together in one way, namely as in the virus helix. The change in relative heights in passing from the disk to the helix is 0.14 nm per subunit and it is conceivable that the packing of the V-columns could not tolerate this much distortion. As a result,9therefore, of packing the V-columns the disk structure may be strained and may attempt to transform into the helix. The contact between the A and B rings of the disk is mediated by a

TIBS -January 1980 LS

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RADIUS (A) Fig. 4 (,4) Schematic view of the inner part of the subunit structure in the virus [13] derived from the electron density maps at 4.0 nm resolution calctdated from X-ray fiber diffraction patterns of the vin~s. A sector of the virus is viewed circumferentially showing the extended LR and RR helices and the V-column joining them together. This part of the stn~aure is not seen in the disk although it is represented by high density in the helical virus. An important component o f the RNA-binding site is the interaction with the LR helix between residues 114 and 123. Th~ part o f the LR helix already exists in tire disk. The triplet of bases (shown black) interacting with the LR helix form a 'claw' grasping tire helix. Two bases are at 4.0 nm radius pointing upwards and one points roughly radially and lies at a larger radius.

complex system of hydrogen bonds which must break when the helix is formed. The disk-helix transition is therefore reminiscent of the R-T transformation in hemoglobin (Durham and Klug [21]). The disk is the low affinity form for RNA because one cannot make the salt bridges to the phosphates without allowing the V-column to build, thereby destabilizing the disk. The helix is the tight binding form. However, to reach the helix it is necessary to break the network of hydrogen bonds between the A and B rings of the disk. The balance between these effects determines the binding of the RNA and makes the RNA binding highly cooperative. Through such cooperativity one could also explain the specificity of the binding. Two properties of the assembly origin seem noteworthy: the requirement that every third residue should be G; and the requirement that the sequence should be long. If we postulate a binding mechanism whereby the selectivity for any one triplet is small (e.g. AGU may have 2 x the binding constant of UUU) but where the simultaneous binding to 16 sites produces the disc-helix transition then one sees at once why the assembly origin can be so specifically recognized - it has the selectivity 2 TM.

Fig. 4 (B) Tire RNA-binding site. The data are taken from [13] and [7~ Shown are tire RNA and tire LR helix. The direction of view is from above with tire center of the virus at tire top o f the diagram. Two repeats are shown. The LR helix ntns above the RNA. In the lefi repeat part of the LR hellr has been omitted for clarity. The bases wh&h rise up out of the plane o f the diagram are shown by ellipses. Tire 2' OH groups are shown with small circles. Note how the 2' OH groups of bases I and 3 come close to Asp- 115 and - 116 o f the LR helix.

Epilogue The above account demonstrates that TMV is a very informative model of prot e i n - R N A interactions. These seem to

proceed by means of a cooperative transition between the disk and the helix involving, amongst other things, the induced folding of a segment of 25 residues. The test lies in the structure. Stubbs and his group in Brandeis are pressing on with the high resolution studies of the virus but for success it will be essential to make use of the detailed information coming out of further studies on the disk structure. It is to be expected that a collaborative enterprise between the Brandeis group and the Cambridge group will soon yield the details of this intriguing structure, thereby fulfilling Rosalind Franklin's dream. References 1 Anderer, F. A. (1963)Adv. Protein Chem. 18, 1 2 Caspar, D. L. D. (1963)Adv. Protein Chem. 18, 37 3 Watson, J. D. (1954) Biochim. Biophys. Acta 13, I0 4 Franklin, R. E. and Holmes, K. C. (1958) Acta Crystallogr. 11,213 5 Franklin, R. E. (1956) Nature (London) 177, 929 6 Finch, J. T., Leberman, R., Chang, Y.-S., and Klug, A. (I 966) Nature (London) 212, 349

7 Bloomer, A. C., Champness, J. N., Bricogne, O., Staden, R. and Klug, A. (1978) Nature (LondotO 276, 362 8 Jack, A. (1973) Acta Crystallogr. A29, 545 9 Bricogne, G. (1976)Acta Co'stallogr. A32,832 10 Klug, A., Crick, F. H. C. and Wyckoff, tt. W. (1958)Acta Co'stallogr. I l, 199 11 Barrett, A. N., Barrington, Leigh J., Ilolmes, K.C., Leberman, R., Mandelkow, E., von Sengbusch, P. and Klug, A. (1971) Cold Spring Harbor Symposia on Quantitative Biology 36,433 12 Holmes, K. C., Stubbs, G. J., Mandelkow, E. and Gallwitz, U. (I 975) Nature (London) 254, 192 13 Stubbs, G. J., Warren, S. G. and Holmes, K. C. (1977) Nature (London) 267, 216 14 Butler, P. J. G. and Klug, A. (1971) Nat, re (New Biol.) 229, 47 15 Butler, P. J. G., Bloomer, A. C., Bricogne, G., Champness, J. N., Graham, J., Guilley, ti., Klug, A. and Zimmern, D. (1976) in Stnlcture-Function Relatiol~hips of Proteins (Markham, R. and Home, R. eds), 3rd John Innes Symposium, p. 101, Elsevier North-Holland, Amsterdam 16 Zimmem, D. and Butler, P. J. G. (1977) Cell l 1, 455 17 Zimmern, D. (1977) Cell 11,463 18 Jonard, G., Richards, K. E., Guilley01t. and Hirth, L. (1977) Cell ! 1,483 19 Butler, P. J. G., Finch, J. T. and Zimmern, D. (1977) Nature (London) 265,217 20 Lebcurier, G., Nicolaieff, A. and Richards, K. E. (1977) Proc. Natl. Acad. Sci. U.S.A. 74, 149 21 Durham, A. C. H. and Klug, A. (1971) Nature (New BioL) 299, 42