J. Mol. Biol. (1990) 212, 247-252

Adenovirus

Serotype 3 Fibre Protein is Expressed as a Trimer in Escherichia coli Corinne Albiges-Rizo and Jadwiga Chroboczek European Molecular c/o I.L.L.,

Biology Laboratory and CXRS, U.R.A. 156X, 38042 Grenoble Cedes-, France

(Received 18 April

1989, accepted 27 November

1333

1989)

The adenovirus serotype 3 (Ad3) fibre has been expressed in Escherichia coli as an insoluble protein. The protein was solubilized by extraction with urea. Slow removal of urea during the purification procedure resulted in a soluble Ad3 fibre preparation. Polyacrylamide gel analysis of the purified fibre protein, as well as cross-linking experiments performed on oellular debris of expressing cells, suggest that the recombinant Ad3 fibre self-assembles as a trimer from identical polypeptide chains. Gel filtration gave the same exclusion volume for the purified recombinant fibre and for the native fibre in the protein mixture extracted from the AdS-infected cells. The recombinant fibre was partially resistant to proteolytic degradation, suggesting a folded structure.

one. Due to the low virulence of Ad3. the concentration of Ad3 fibre in the infected cell is very low making fibre purification difficult’. We have, therefore, undertaken the expression cloning of the Ad3 fibre in Escherichia coli with the aim of obtaining material for structural st,udies. We have used a gene expression system based on bact’eriophage T7 polymerase (Studier & Moffatt, 1986). As a result of cloning of the coding region of Ad3 fibre we obtained a recombinant. plasmid whose translation product should be a fusion protein consisting of the first 11 N-terminal amino acids of phage T7 protein 10, three amino acids derived from the linker sequence and 319 amino acids specified by the sequence of Ad3 fibre. When total protein extracted from expressing cells was analysed on SDS/polyacrylamide gel, a band was visible at an M, of about 36,000, corresponding to the expected position for Ad3 fibre (Fig. l(a)). The successful expression of the Ad3 fibre protein was confirmed by Western blotting (Fig. l(b)). The recombinant fibre seems to be stable during thr course of expression in pulse and chase experiments (results not shown). The expressed protein was insoluble and could be solubilized either bv denat,urants such as urea above 2M or 4M-guanidinium hydrochloride. or in buffers above pH Iti7 or below pH 1.X. Elenatron microscopy of thin cell sections prepared from expressing cells revealed the presence of inclusion bodies, which were absent in non-expressing cells (results not shown). When Western blot analysis of cellular debris was performed with the overloaded gel. the results of

The attachment of adenovirus to the host cell is mediated by elongated proteins (fibres) protruding from the vert,ices of the virion. Electron microscopy of Ad2t ,and Ad5 fibre has revealed that it consists of a shaft with a terminal knob (Mautner & Pereira, 1971: Devaux rt al., 1984). Structural studies on the Ad2 fibre have suggested that this molecule is either a dimer or a trimer. In the study by Green et al. (1983), a model in favour of a dimeric structure of t,he Ad2 fibre has been proposed in which the shaft portion of the fibre is composed of two parallel cross-p structures. However, Van Oostrum & Burnett (1985) deduced from stoichiometric analysis that the Ad2 fibre is trimeric, with the monomer molecular weight of 61,925. By sequence analysis of the gene it has been shown that the fibre polypeptide of another adenovirus, serotype 3 (Ad3) consists of 319 amino acid residues, corresponding to a molecular weight of 34,800 (Sign&s et al., 1985). Due t’o the scarcity of native Ad3 fibre, nothing is known about, the higher-order structure of this protein. Comparison of the Ad2, Ad5 and Ad3 fibre protein sequences shows the conservation of some amino acids, such as glycine and proline, which in both sequences are found in quasi-periodic positions (Chroboczek & Jacrot, 1987). These are possibly the key positions necessary for maintaining the higherorder structure of these proteins (Green et al., 1983), which suggested that the native Ad3 fibre should also have the form of a trimer, possibly a shorter tXbbrrviat,ion used: 4d%, Ad3, 4d5, adenovirus srrotypr 51.3. 5. ~~2%2836/90/06X1247+06

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1990 Acadruric

Press Limited

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Figure 1. Expression of Ad3 fibre protein in ,!C’.coli. Cells grown in LB medium were induced with ti4 mM-isopropyl ,8-o-t,hiogalectopyranoside. The crlls were pelleted and boiled in Laemmli sample buffer (Laemmli, 1970). The samples were run on an SD8/12.5% polyacrylamide gel. Proteins were stained with (a) Coomassie brilliant’ blue and (b) recombinant Ad3 fibre protein was revealed with an anti-Ad3 serum. Lane 1, E. co& BL21(DE3) containing plasmid with Ad3 fibre gene inserted in a wrong reading frame. before induction: lane 2. same cells. after induction: lane 3. K. coli BL21(DE3) containing plasmid with fibre gene inserted in a caorrect reading frame. before induction: lane 4. same cells, aft,er induction. Lane M. molecular weight markers. The position of recombinant Ad3 fibre protein is marked with a dot. (c) Purification of Ad3 fibre protein synthetized in IY:. coli. Samples from various stages of the purification were analysed on an SDS/lSUb polyacrylamide gel st,ained with C’oomassir brilliant blue. Lane 1, total ~11 extract: lane 2. soluble fraction: lane 3. supernatant after extraction of the pellet with 6 Al-urea: lane 4. pooled fraction from Q-Sepharosr: lane 5. pooled fraction from Blur-Sepharosr: lane 6. molecular weight markers. The position of the recaombinant protein is indicated by an arro\+

31,000

45,000

66,200

66,200

2

3

4

5

6

Figure 3. Proteolysis of rrc:ornbimu~t~ Ad3 fibre protein. Hydrolyses were performed [lanes 3). 20 min (lanes 4). 30 min (lanes 5) and 60 min (lanes 6). respectively. with a ratio

I

2

3

(b)

4

5

6

at 20°C‘ wit,h (a) trypsin and (b) ehymot~rypsill for 5 min (lanes 2). 10 milr of enzyme to substrate of 1 : 200. Lanes 1, undigested Ad3 recombinant fibrr

I

Communications immunodetection were quite complex, since besides the 36,000 M, band two prominent bands with lower mobility (apparent M,72,000 and 100,000) could be seen, probably representing the different states of oligomerization of the expressed protein. Also visible were polypeptide chains of smaller molecular mass than the Ad3 fibre monomer, which might be proteolytic breakdown products or premature termination products. The polypeptide chain of Ad3 fibre contains four cysteine residues with the potential to form intramolecular or intermolecular disulphide bonds. However, heating recombinant protein in the presence of different concentrations of dithiothreitol and /?-mercaptoethanol left intact the two bands with high molecular weight. These three bands were tentitavely identified as the monomer, dimer and t)rimer of the fibre according to their apparent molecular weights on SDS/polyacrylamide gel. In order to test this hypothesis we treated the cell debris obtained after lysis with the reagent dimethyl suberimidate, a bifunctional agent that can form cross-links between lysine residues (Davies & Stark, 1970; Gething et al., 1986; Fig. 2, lane 1). The same three major bands were observed but the minor bands disappeared when recombinant protein was cross-linked. These results suggest that the minor bands seen in the absence of dimethyl suberimidate could represent different forms of protein partially unfolded by SDS. Similarly, the different oligomeric species have been observed for the trimeric transmembrane protein, porin, and it has been postulated that both electrostatic and hydrophobic interactions contribute to the stability of this protein (Schindler & Rosenbusch, 1984; Rocque et al., 1987). When a purified preparation of recombinant prot,ein was run without cross-linking on a denat’uring gel, the same three predominant species could be seen (Fig. 2(b)). It is worth noting that in this case the amount of trimer exceeded that’ of dimer. Under non-denaturing conditions only one protein species could be seen on a polyacrylamide gel (Fig. 2(c)). It’ shows that after removal of the urea the recombinant Ad3 fibre exists as a highly homogeneous population of molecules, which we interpret, as a trimeric fibre protein. This preparation was used for the generation of antibodies in rabbits. Gel filtration on Sephacryl S-300 was used for the comparison of the exclusion volume of purified recombinant protein with that of native fibre protein contained in the extract of Ad3-infected KB cells. The presence of fibre protein was revealed by anti-recombinant protein serum. Native fibre protein was excluded at nearly the same volume as the recombinant one: 55 to 57 ml versus 54 to 57 ml. These results strongly suggest that the protein produced in E. coli and nat’ive fibre are in the same st.at,e of oligomerization. Partial proteolysis can be used as an indicator of protein conformation (Fisher & Blumenthal, 1980). In the case of Ad2 fibre protein, chymotrypsin

251

digestion leads initially to the conversion of the into one of 60,000. This 62,000 M, polypeptide cleavage occurs at tyrosine 16 of the Ad2 polypeptide chain (Devaux et al., 1987). From the pronounced homology of the sequence of Ad2 and Ad3 fibres at the N terminus, we could expect a similar cut at tyrosine 16 (which in the case of our fusion protein is tyrosine 29). Assuming that the Ad3 fibre protein has a compact struct’ure, which similarly to Ad2 fibre results in a low sensitivity to proteolysis, one could expect the change of molecular mass of our fusion protein from 36,000 to 33,000 Da. The results shown in Figure 3(b) are consistent with this hypothesis. Digestion with trypsin led to the conversion of the 36,000 M, polypeptide chain into two bands of 32,000 and 26,000, respectively (Fig. 3(a)). The action of these two enzymes seems to be similar, in the sense that the peptides in the same range of molecular weight are formed with similar kinetics. It’ seems that once the principal cuts are made, the structure of whole protein becomes more open and further proteolysis results probably in the formation of small peptides. From t.he electrophoret.ic behavionr of purified recombinant fibre as well as from t,he results of cross-linking experiments it’ seems t’hat the rraombinant protein is self-assembled into a t,rimer. During gel filtration, recombinant Ad3 fibre is excluded at the same volume as the viral protein, which suggests t’he same level of oligomerizat)ion and possibly folding for both proteins. Preliminary neutron scattering experiments performed with the solution of the purified protein indicate an M, of 100,000 to 120.000 (I’. Timmins, personal communication), which is again consistent. wit,h a. trimeric structure for the recombinant protein. Bearing in mind that the neutron scattering data are not subject to the same artifacts as cross-linking and gel filtration techniques, all these results point to the fact that Ad3 fibre is a trimer, similar bo the Ad2 tibre. It is wort’h noting that in the case of Ad3 fibre expression the higher-order structure (trimer) of this rukarvotic protein appears to be formed in a prokaryo& cell, in the absence of any other viral product. It implies that the folding and trimerizat’ion of fibre during the viral life cycle probable proceeds simply t’hrough self-assembly. This is unlike t.he hrxon, the major adenovirus structural protein. whose assembly requires t.he action of an rarlv viral protein, which forms a tight complex with hexon polypeptides (Cepko bz Sharp, 1982). Our experiments indicate that the recombinant Ad3 fibre protein acquires a folded structure upon expression and purification but the extent’ of proper folding is not known, particularly as the native virus-produced protein has not yet been purified. The scarcity of the fibre protein isolated from Ad3infected cells has severely limited st)udies on the structure of this protein. We expect t)his recombina,nt. Ad3 fibre and the antibodies against it to be useful in studying the struct.ure and the function of Ad3 fibre protein.

252

(‘. Abiges-Rim

We warmly acknowledge our debt to K. Jacrot who initiated this st,udy. We thank W. Studier for providing the expression system, 11. Petterson for the pAd3 plasmid containing the fibre gene and (1. Devaux for providing antibodies against total Ad3. We are grateful to M. Adrian for electron microscopy and to P. Timmins for performing neutron scattering experiments. We thank P. Neuner for oligonucleotide synthesis. We are also grateful to S. Cusack for thoughtful comments on the manuscript.

and J. Chroboczek Devaux. (‘.. (‘aillet,-Boudin. M. I,.. Jacrot. 1% KBoulanger. P. (1987). Virology, 161. 161-128. Fisher. R. & Blumenthal. ‘I’. (1980). J. Biol. Ch.mr. 255. 1105ci-11062.

Get’hing, M.. McCa.mmon, K. & Sambrook. J (1986). C’rll. 46, 939-950. Green. N. ?ul..Wrigley, N. (i.. Russel. b$‘. (‘.. &lartin, S. R. & McLachlan. A. 1). (1983). E,!4HO J. 8. 1357 -1365. Laemmli. C. K. (1970). Nature (London). 227, 680--68.5 Mautner. V. & Pereira. H. G. (1971). Xatzm (London/, 230. 4.56.--457.

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Rocque. W. ,I.. c‘ouglin, K. ‘I’. & Mc(:roattbr. E. -1. (1987). ,J. Ba.ctrriot. 169. 40044010. Scbhindler, M. & Rosenbuch. .J. 1’. (1981). E’EHS Lrfl~rs.

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