The transmembrane oligomers of coronavirus protein E

Abstract

We have tested the hypothesis that severe acute respiratory syndrome (SARS) coronavirus protein E (SCoVE) and its homologs in other coronaviruses associate through their putative transmembrane domain to form homooligomeric alpha-helical bundles in vivo. For this purpose, we have analyzed the results of molecular dynamics simulations where all possible conformational and aggregational space was systematically explored. Two main assumptions were considered; the first is that protein E contains one transmembrane alpha-helical domain, with its N- and C-termini located in opposite faces of the lipid bilayer. The second is that protein E forms the same type of transmembrane oligomer and with identical backbone structure in different coronaviruses. The models arising from the molecular dynamics simulations were tested for evolutionary conservation using 13 coronavirus protein E homologous sequences. It is extremely unlikely that if any of our assumptions were not correct we would find a persistent structure for all the sequences tested. We show that a low energy dimeric, trimeric and two pentameric models appear to be conserved through evolution, and are therefore likely to be present in vivo. In support of this, we have observed only dimeric, trimeric, and pentameric aggregates for the synthetic transmembrane domain of SARS protein E in SDS. The models obtained point to residues essential for protein E oligomerization in the life cycle of the SARS virus, specifically N15. In addition, these results strongly support a general model where transmembrane domains transiently adopt many aggregation states necessary for function.

FIGURE 1

Complete sequence of SCoVE. The predicted TME used in the simulations is indicated (shaded bar). The corresponding transmembrane sequence used for other variants is shown in the alignment of Fig. 2. Three cysteines (black circles) C40, C43, and C44 are indicated, which are possible palmitoylation sites.

FIGURE 2

Sequences corresponding to the putative transmembrane segments of SARS coronavirus E protein and its homologous used in our molecular dynamics simulations. The column on the left indicates their abbreviated name. The complete name and corresponding Swiss-Prot entries are indicated in the Materials and Methods section. The numbering corresponds to SCoVE. The residue used to calculate the rotational orientation, ω₂₃, for the models in Figs. 3–5 is indicated by an asterisk.

FIGURE 3

(a) Plot of helix tilt versus ω₂₃ for the low energy models (each symbol represents one model) obtained after the GSMD simulations for a homodimeric model when restraining the helix tilt to 10°. For each sequence, the horizontal broken line separates left-handed (symbols above the broken line) from right-handed bundles (symbols below the broken line). The vertical broken line indicates the average orientation (at ω = −23°) where the complete set was found (RMSD, 1.5 Å; n = 10 structures). The models inside the small rectangles are those forming a complete set. (b) The models in panel a are represented as a function of their energy (ordinate axis) and ω₂₃. The lowest energy models found in each sequence are indicated with a shaded rectangle.

FIGURE 4

As in Fig. 3, but assuming a homotrimeric homooligomer. This figure only shows the results when the helix tilt was restrained to 35°. The vertical broken line indicates the orientation at ω = −113, where the complete set was found (RMSD, 1 Å; n = 10).

FIGURE 5

As in Fig. 3, but assuming a homopentameric homooligomer. Only restraining the helix tilt to 25° (shown here), a complete set was found (RMSD, 1Å; n = 10). The vertical broken lines indicate the orientation of the complete sets found, at ω = −121° (form A) and at ω = −176° (form B).

FIGURE 6

Columns from left to right: slices through the dimeric, trimeric, pentameric-form A and pentameric-form B of the transmembrane domain of SARS coronavirus E protein, i.e., sequence SCoVE in Fig. 2. Color code: L, green; V, cyan; I, salmon; A, marine; F, blue; N, orange; and S and T, red. For clarity's sake, the residue numbers are indicated only in one of the helices of the trimeric model. Note the central role of N15 for the three types of oligomers.

FIGURE 7

SDS-PAGE electrophoresis corresponding to the synthetic transmembrane peptide of SARS protein E. Lane 1 (left) shows the molecular weight markers. Lanes from 2 to 4: increasing load of peptide: 10, 20, and 40 μg, respectively. Arrows indicate the bands corresponding to the dimer, trimer, and pentameric forms of the peptide. The bands corresponding to the pentamer in lanes 2 and 3 were visible only after silver staining.