key: cord-0949483-5952zazu
authors: de Groot, R. J.; Luytjes, W.; Horzinek, M. C.; van der Zeijst, B.A.M.; Spaan, W.J.M.; Lenstra, J. A.
title: Evidence for a coiled-coil structure in the spike proteins of coronaviruses
date: 1987-08-20
journal: Journal of Molecular Biology
DOI: 10.1016/0022-2836(87)90422-0
sha: 69820b60dc25dcca180ae127af70141e8b8ae16f
doc_id: 949483
cord_uid: 5952zazu

Abstract The amino acid sequences of the spike proteins from three distantly related coronaviruses have been deduced from cDNA sequences. In the C-terminal half, an homology of about 30% was found, while there was no detectable sequence conservation in the N-terminal regions. Hydrophobic “heptad” repeat patterns indicated the presence of two α-helices with predicted lengths of 100 and 50 Å, respectively. It is suggested that, in the spike oligomer. these α-helices form a complex coiled-coil, resembling the supersecondary structures in two other elongated membrane proteins, the haemagglutinin of influenza virus and the variable surface glycoprotein of trypanosomes.

. I. J!lol. Rid. (1987) 1%. 963-966 Evidence for a Coiled-coil Structure in the Spike Proteins of Coronaviruses

The amino acid sequences of the spike proteins from three distantly related coronaviruses have been deduced from cDNA sequences. In the C-terminal half, an homology of about 3Oqb was found, while there was no detectable sequence conservation in t'he N-terminal regions. Hydrophobic "heptad" repeat patterns indicated the presence of two cr-helices witjh predicted lengths of 100 and 50 A, respectively.

It is suggested that, in the spike oligomer. these cl-helices form a complex coiled-coil, resembling the supersecondary structures in two other elongated membrane proteins, the haemagglutinin of influenza virus and the variable surface glycoprotein of trypanosomes.

Coronaviruses are enveloped RNA viruses with a single-stranded genome of positive polarity (Siddell et al., 1983; Sturman & Holmes, 1983) . They cause considerable economical damage by infecting livestock and other domestic animals. Projecting from their surface are unusually large (-200 A), petalshaped spikes. These so-called peplomers mediate the binding of virions to the host cell receptor and are involved in membrane fusion. Further, they are considered the main targets of the protective immune response (Sturman & Holmes, 1983; Cavanagh et al.. 1986a) .

Each peplomer consists of a dimer or possibly a t)rimer of the peplomer protein (Cavanagh, 1983) , a glycoprotein of 180,000 to 210,000 M, (Sturman & Holmes. 1983; Jacobs ut al., 1986; Boyle et al., 1984 A stretch of 20 to 25 hydrophobic residues, found near the C terminus, most probably serves as a transmembrane anchor. Amino acid sequences have been aligned by the following procedure. Initial amino acid alignments were obt,ained by FASTP analysis (Lipman & Pearson, 1985) . These alignments have been extended by reiterating FASTP with non-aligned parts as query sequence and by DTAGON cornparison ( Fig. l(a) ; Staden, 1982) . The results are summarized in Figure 2 . Most' conservation is observed in the C-terminal half of the proteins. with overall amino acid homologies of 35, 30 and SS(!, for TBV-FTPV, TBV-MHV and MHV-FTPV, respectively: about 50% of t'he amino acid substit,utions may be considered conservative (Dayhoff of al., 1983) . Tn contrast, we did not, find significant homology or matching cysteine residues in the N-terminal segments; amino acid residues t,hat could be aligned by introducing numerous gaps were not conserved in closely related strains of IR\ (Niesters, 1987) or MHV (Luytjcs rf ~1.. unpublished results). Furthermore. insertions or deletions in the N-terminal domains ac*count largeI! for the differences in size of the prplornrr apoproteins.

No experimental data are available on t.he structure of the peplomers. However. I)TA(X)N plots revealed two repetit)ious regions in the C-terminal domains wit,h a seven-residue periodicity (Fig. l(b) ). Closer analysis showed the presence of so-called "heptad repeats" (Cohen Cyr Parrv, 1986 ). i.e. a sequence periodicity (a-1)-c-d-e-f-g) in whit+ the residues in the a and d posit,ionx gcbneralty are hydrophobic (Fig. 3) . Statistical tests of t,he predominant occurrence of hydrophobic2 residues in the a and d positions yielded confidence levels of at least 962;(,; in the long repetitive regions, t,hr two parts with different, heptad phasings have been tested separately.

Heptad repeats are indicative of' a coiled-coil structure in which the hydrophobic~ residues form the interface between interlocking a-helixes (Cohen B Parry, 1986 ). Tn accordance with the presumptive a-helical conformation, the repeats in the peplomer proteins are located in regions devoid of helix-breaking proline residues.

For the minor repeat near the transmembrane anchor (Figs 2 and 3 ) an a-helix of 50 (MHV and IBV) or 70 A (FIPV) may be predicted. The major repeat indicates a helix of at least 100 (TBV and MHV) or 130 A (FTPV), spanning more than half the peplomer. Note that in FIPV the minor and major repeats contain one insertion of 21 residues and two insertions of seven residues, respectively; thus three and two heptads are added, while the repeat pattern is conserved.

The presence of two heptad repeats suggests an int,ra-chain coiled-coil. However, this would leave FIPV about 50 A of the predicted major helix unpaired. Therefore, it is assumed that in the oligomer the major helices are involved also in an inter-chain coiled-coil. Such a structure would resemble the complex coiled-coils found in the dimeric variable surface glycoproteins (VSG) of t'rypanosomes (Metcalf et al., 1987) and the haemagglutinin trimer (HA) of influenza virus (Wilson et al., 1981) . Tn these proteins, bundles of four (VSG) or three (HA) a-helices with lengths of 90 and 76 A, respectively, are surrounded by shorter helices; the interaction of the long helices stabilizes the oligomer. The influenza virus HA and the coronavirus peplomer are functionally analogous, both carrying the receptor binding site and mediating membrane fusion. We propose that, t'hese surface projections have converged to a similar super-secondary structure in order to position the receptor binding site at some distance from the membrane. Thus, the typical elongat.ed shape of the coronavirus peplomer may be explained by a model (Fig. 4) , in which a coiled-coil with a predicted length of 100 to 130 AA forms t,hr connection between the globular 1 Membrane Figure 4 . Tentative model of' the coronavirus peplomer. The peplomer is represented as a dimer. The transmembrane cc-helicrs and the cc-helices in the coiledcwil structure are depicted as rounded cylinders. part and the viral membrane.

As in HA (Wilson rt al., 1981) , the protein surface near the membrane may carry carbohydrate groups. att)ached to potential glycosylation sites in the region ('ontaining the minor heptad repeat (Fig. 2) . The bulbous part of the peplomer prot,ein probably contains the non-conserved X-terminal seclucww ( Fig. 2 

Institute of L'irology and ' Department of Bacteriology. Veterinary Faculty. L7nivrrsity of 1Ttrrcaht. Yalelaan I 3.584 ("I, I'trerht. The Netherlands Received 2

& Wile?;. 1). C. (1987). Nature (London)

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