Structure of a conserved Golgi complex-targeting signal in coronavirus envelope proteins

Abstract

Coronavirus envelope (CoV E) proteins are ∼100-residue polypeptides with at least one channel-forming α-helical transmembrane (TM) domain. The extramembrane C-terminal tail contains a completely conserved proline, at the center of a predicted β-coil-β motif. This hydrophobic motif has been reported to constitute a Golgi-targeting signal or a second TM domain. However, no structural data for this or other extramembrane domains in CoV E proteins is available. Herein, we show that the E protein in the severe acute respiratory syndrome virus has only one TM domain in micelles, whereas the predicted β-coil-β motif forms a short membrane-bound α-helix connected by a disordered loop to the TM domain. However, complementary results suggest that this motif is potentially poised for conformational change or in dynamic exchange with other conformations.

Keywords: Analytical Ultracentrifugation; Coronavirus; Envelope Protein; Golgi Targeting; Nuclear Magnetic Resonance (NMR); Protein Structure; SARS; Structural Biology; Viral Protein; Virus Assembly.

FIGURE 1.

Sequences, expression, and purification of SARS-CoV E_TR. A, alignment of representative sequences of E proteins in α-, β-, and γ-coronaviruses. The cysteine residues are underlined, the conserved proline is highlighted (gray), and the four residues mutated to alanine in the E_4ALA mutant (see “Materials and Methods”) are shown in red. For these four proteins, the prediction of secondary structure is shown below in a color code, with the TM domain indicated as a black line; B, proteins used in the present work: a His-tagged construct (E_TR) encompassing residues 8–65 (boldface type, underlined), and full-length SARS-CoV E (E_FL). In E_FL, the fragment SNA results from the cleavage of the tag. In both proteins, the native cysteines were mutated to alanine (C40A, C43A, and C44A; see asterisks); C and D, MALDI-TOF MS spectra (C) and standard SDS-PAGE (D) of pure E_FL with the species labeled; E and F, same as for purified E_TR; the identities of various single- and double-charged species are indicated. The calculated mass of E_TR is 8,995 Da.

FIGURE 2.

Topology and secondary structure of E_TR. ¹H-¹⁵N TROSY-HSQC spectra of 0.2 mm E_TR in 50 mm SDS in H₂O (A) and in 99% D₂O (B). The cross-peaks are labeled by one-letter code and residue number; C and D, peak intensity reduction upon the addition of 5-DSA (C) and 16-DSA (D), calculated as the ratio of peak intensity before and after the addition of the paramagnetic reagents; E, ¹H-¹⁵N steady-state heteronuclear NOE experiment; F, sequential and medium-ranged NOE connectivity between residues, displayed as bands under the respective residues.

FIGURE 3.

Structural model of E_TR. A, superposition of an ensemble of 15 calculated simulated annealing structures of E_TR (only the sequence corresponding to E protein, 8–65, is shown). Side chains are shown as line representations; the residues at the ends of the two helical segments are indicated. B, residues of the C-terminal extramembrane α-helix oriented toward the micelle surface (blue). C, ribbon representation of the TM central region, with the carbonyl oxygen of Phe-26 forming a hydrogen bond to the side chain of Thr-30.

FIGURE 4.

Equivalence in secondary structure of E_TR and E_FL. A, CD spectra of E_TR in DPC (black), 1:2 molar ratio SDS/DPC mixture (blue), and SDS (red). B, infrared amide I band of E_TR (red) and E_FL (blue) in DMPC lipid bilayers and their respective Fourier self-deconvolved spectra (dotted lines). C, comparison of secondary ¹³Cα chemical shifts (deviation from tabulated random coil ¹³Cα chemical shift values) for E_TR (red dots) and E_FL (blue dots) in SDS micelles and for E_TR in (1:4 molar ratio) mixed SDS/DPC micelles (white dots). For the latter, Pro-54 and Thr-55 were excluded from the analysis due to significant line broadening; Arg-38 was excluded from the analysis due to the peak overlapping.

FIGURE 5.

Channel activity of E_TR and interaction with HMA. A, I/V plot for E_TR in 1,2-diphytanoyl-sn-glycero-3-phosphocholine bilayers in a symmetrical experiment where both cis and trans compartments contained 10 mm HEPES and 500 mm NaCl at pH 5.5. Each point represents the mean of at least three current readings. The line is a linear regression fit of data points, which produced a slope of 0.39 ± 0.02 nanosiemens. B, selected traces of 12 s each, recorded at various holding potentials of E_TR. C, channel activity recorded at 60 mV holding potential and after the addition of 10 μm HMA (arrow). D, ¹H-¹⁵N TROSY-HSQC spectra of 0.1 mm ¹⁵N-labeled E_TR in SDS micelles (D) before (blue) and after (red) the addition of 1 mm HMA. E, same as for 0.2 mm ¹⁵N-labeled E_TR in (1:4 molar ratio) SDS/DPC micelles upon titration with 0.4 mm HMA. Some shifts are indicated with arrows; F, CSP of the backbone amide resonances of E_TR before and after the addition of HMA in SDS (red) and (1:4 molar ratio) SDS/DPC micelles (blue). Note that the HMA/E_TR molar ratio was 10 in SDS and only 2 in SDS/DPC micelles. The arrows show residues with significant change in chemical shifts after the addition of HMA. The TM domain is indicated only to guide the eye.

FIGURE 6.

Gel electrophoresis of E_TR in SDS and in a SDS/DPC mixture. Shown are E_TR in 4–12% SDS-NuPAGE (A) and 4–16% blue native PAGE (B) of E_TR in 25 mm SDS with increasing concentration of DPC, as indicated. E. coli aquaporin Z (AQPZ) was included as an additional molecular weight marker. Bands and oligomeric states are indicated by arrows and black dots, respectively.

FIGURE 7.

Sedimentation equilibrium of E_TR in SDS/DPC micelles. A, radial distribution profiles (open circles) of E_TR in a (25:100 mm) SDS/DPC mixture at 16,600 rpm (red), 20,300 rpm (green), and 24,900 rpm (blue). The profile was fitted to a monomer-pentamer self-association model (black line), and the fitting residuals are shown below each plot. B–D, global reduced χ² values obtained after data fits to different monomer/n-mer models of E_TR association in SDS/DPC micelles (B), a 50:100 mm SDS/DPC mixture (C), and 5 mm C14-betaine (D). E, 4–12% PFO-NuPAGE of E_TR.

FIGURE 8.

Interaction between E_TR and Nsp3a. A, sensorgrams corresponding to the interaction between purified RSV SH(45–65) and immobilized E_TR (red). The steady-state model (dark red) did not produce a good fit to a 1:1 model of interaction. The association phase extends from 0 to 60 s, whereas the dissociation phase extends for minutes. B, same as for Nsp3a and immobilized E_TR (blue) and fit to a steady-state model (red). Inset, dose-response plot, where the equilibrium responses of Nsp3a were plotted against the log₁₀(concentration) of Nsp3a. Although the fit (blue line) yields an affinity of 1.6 mm, binding is already evident even in the interval 1–10 μm Nsp3a concentration. C, CSP of the backbone amide resonances of 0.2 mm ¹⁵N-labeled E_TR in (1:4 molar ratio) SDS/DPC micelles upon titration with 0.4 mm Nsp3a (blue dots) or the negative control RSV SH(45–65) (red dots).

FIGURE 9.

Structural flexibility at the putative β-coil-β motif. A, amide I band corresponding to E_FL, E_P54A, and E_4ALA in DMPC bilayers. Regions that change after mutation of the four residues indicated in Fig. 1A (E_4ALA) are shown as arrows. B, Fourier self-deconvolved spectra corresponding to the amide I bands shown in A. C, infrared amide I and II bands corresponding to SARS-CoV E peptide E_46–60 in DMPC lipid bilayers before (blue) and after (red) being exposed to D₂O (39). D, possible equilibrium between two conformations at the putative β-coil-β motif, shifted toward an α-helical form, between the model determined experimentally for E_TR and E_FL and one built with prediction tools (PEP FOLD) (3) and consistent with data obtained with synthetic fragment E_46–60 shown in C.

FIGURE 10.

Pentameric model formed by E_TR. Side (A) and top (B) views of a proposed E_TR pentamer structure shown in ribbon representations. The side chains of Val-49 and Leu-65, which have been shown to interact with HMA, are shown as line representations.