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. 2014 May 2;289(18):12535-49.
doi: 10.1074/jbc.M114.560094. Epub 2014 Mar 25.

Structure of a Conserved Golgi Complex-Targeting Signal in Coronavirus Envelope Proteins

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Free PMC article

Structure of a Conserved Golgi Complex-Targeting Signal in Coronavirus Envelope Proteins

Yan Li et al. J Biol Chem. .
Free PMC article

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.

Figures

FIGURE 1.
FIGURE 1.
Sequences, expression, and purification of SARS-CoV ETR. 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 E4ALA 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 (ETR) encompassing residues 8–65 (boldface type, underlined), and full-length SARS-CoV E (EFL). In EFL, 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 EFL with the species labeled; E and F, same as for purified ETR; the identities of various single- and double-charged species are indicated. The calculated mass of ETR is 8,995 Da.
FIGURE 2.
FIGURE 2.
Topology and secondary structure of ETR. 1H-15N TROSY-HSQC spectra of 0.2 mm ETR 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.
FIGURE 3.
Structural model of ETR. A, superposition of an ensemble of 15 calculated simulated annealing structures of ETR (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.
FIGURE 4.
Equivalence in secondary structure of ETR and EFL. A, CD spectra of ETR in DPC (black), 1:2 molar ratio SDS/DPC mixture (blue), and SDS (red). B, infrared amide I band of ETR (red) and EFL (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 ETR (red dots) and EFL (blue dots) in SDS micelles and for ETR 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.
FIGURE 5.
Channel activity of ETR and interaction with HMA. A, I/V plot for ETR 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 ETR. 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 ETR in SDS micelles (D) before (blue) and after (red) the addition of 1 mm HMA. E, same as for 0.2 mm 15N-labeled ETR 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 ETR before and after the addition of HMA in SDS (red) and (1:4 molar ratio) SDS/DPC micelles (blue). Note that the HMA/ETR 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.
FIGURE 6.
Gel electrophoresis of ETR in SDS and in a SDS/DPC mixture. Shown are ETR in 4–12% SDS-NuPAGE (A) and 4–16% blue native PAGE (B) of ETR 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.
FIGURE 7.
Sedimentation equilibrium of ETR in SDS/DPC micelles. A, radial distribution profiles (open circles) of ETR 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 ETR 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 ETR.
FIGURE 8.
FIGURE 8.
Interaction between ETR and Nsp3a. A, sensorgrams corresponding to the interaction between purified RSV SH(45–65) and immobilized ETR (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 ETR (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 ETR 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.
FIGURE 9.
Structural flexibility at the putative β-coil-β motif. A, amide I band corresponding to EFL, EP54A, and E4ALA in DMPC bilayers. Regions that change after mutation of the four residues indicated in Fig. 1A (E4ALA) 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 E46–60 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 ETR and EFL and one built with prediction tools (PEP FOLD) (3) and consistent with data obtained with synthetic fragment E46–60 shown in C.
FIGURE 10.
FIGURE 10.
Pentameric model formed by ETR. Side (A) and top (B) views of a proposed ETR 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.

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