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

Background: Coronavirus envelope (CoV E) proteins have a predicted β-coil-β motif reported to target the Golgi complex.

Results: This conserved domain forms β-structure on its own but is α-helical in the context of full-length SARS-CoV E protein.

Conclusion: This domain is potentially involved in large conformational transitions.

Significance: This is the first structural data of the extramembrane domain of any coronavirus E protein.

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.

Introduction

Coronaviruses (CoV³; order Nidovirales, family Coronaviridae, subfamily Coronavirinae) are enveloped viruses organized into three groups (1, 2)⁴: group 1 (α-coronaviruses), group 2 (β-coronaviruses) and group 3 (γ-coronaviruses). Coronaviruses have been known to cause common cold symptoms in humans and a variety of lethal diseases in birds and mammals (4). However, in 2003, the virus responsible for the severe acute respiratory syndrome (SARS-CoV) (5) produced a near pandemic with 8,273 cases and 775 deaths (6). In 2012, a novel β-coronavirus (HCoV-EMC) (7–9) was discovered that has already led to many fatalities (10–12).

The main coronavirus structural proteins are S (spike), E (envelope), M (membrane), and N (nucleocapsid), where S, E, and M are integral membrane proteins. E proteins are ∼100-residue-long polypeptides that are minor components in virions but are abundantly expressed inside infected cells (13). They have a short hydrophilic N terminus, at least one predicted terminal transmembrane (TM) domain, and a less hydrophobic C-terminal tail.

Co-expression of E and M proteins is sufficient for formation and release of virus-like particles (14–19), and E proteins have been proposed to participate in inducing membrane curvature or in the scission of particles (20). Mutations at their C-terminal extramembrane domain impair viral assembly and maturation in the murine hepatitis virus (20). In the transmissible gastroenteritis virus, the absence of E protein resulted in a blockade of virus trafficking in the secretory pathway and prevention of virus maturation (21, 22). In the case of the severe acute respiratory syndrome coronavirus (SARS-CoV), viruses lacking the E gene showed attenuation and did not grow in the central nervous system (23, 24), whereas stress response genes were up-regulated and cell apoptosis increased (25). These results suggested a role of E protein in both tissue tropism and pathogenicity, where modulation of stress responses contributes to viral attenuation. In fact, SARS-CoV ΔE attenuated viruses constitute promising vaccine candidates (26–28). Thus, although E proteins are not absolutely essential for in vitro or in vivo coronavirus replication, their absence is clearly deleterious.

Immunofluorescence experiments have shown that SARS-CoV E has a cytoplasmically oriented C terminus and a lumenal N terminus (i.e. an N_exoC_cyto orientation) (13), which is consistent with the presence of a single TM domain. SARS-CoV E protein and other CoV E proteins have channel activity in synthetic membranes (29–31). This channel activity is mediated by formation of pentameric oligomers (32–34) and is only very mildly selective for cations (35). The only available structural data for CoV E proteins, obtained using synthetic TM peptides, is derived from the channel-forming TM domain in SARS-CoV E (32, 34, 36). No structural data are available for the predicted N- or C-terminal extramembrane domains despite the latter being critical for viral assembly (37, 38), although some results have been obtained using shorter synthetic peptides encompassing this domain (39). The C-terminal domain of E proteins contains a totally conserved proline residue, which in β- and γ-coronaviruses is at the center of a predicted β-coil-β motif (Fig. 1A), reminiscent of viral internal fusion peptides (40–42). In SARS-CoV E, this motif was found to be responsible for redirecting a plasma membrane protein to the Golgi region. Conversely, mutations designed to increase its α-helical propensity disrupted localization to membranes (43).

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.

The C-terminal tail of E proteins is also important for its interaction with the C-terminal domain of M protein (18, 44, 45) at the cytoplasmic side of the endoplasmic reticulum-Golgi intermediate compartment, the budding compartment of the host cell. These interactions are the major drivers for envelope formation (46). The C-terminal tail of SARS-CoV E protein also interacts with the cellular protein PALS1 (47), which is thus depleted from the tight junctions in epithelial cells. Finally, SARS-CoV E interacts with the seven-domain (48) SARS non-structural protein 3 (Nsp3) (49).

Thus, it is crucial to determine the structure of the C-terminal predicted extramembrane domain of E proteins, especially in the context of a large construct that includes the TM domain. Until now, these structural studies have been hampered by problems in expression, purification, and stabilization of E proteins. We report herein the first detailed structure of a truncated form of the SARS-CoV E monomer that includes both its TM domain and its predicted β-coil-β motif at the C-terminal tail.

MATERIALS AND METHODS

SARS CoV E Protein Constructs

Full-length SARS CoV E and a truncated version were used in this work. Full-length SARS-CoV E protein (E_FL) was cloned into pTBMalE plasmid downstream of the MBP fusion tag and tobacco etch virus cleavage sequence. The plasmid was transformed into Escherichia coli strain BL21(DE3) codon plus for protein expression. The truncated form, E_TR, was cloned into pNIC28-Bsa4 with an N-terminal His₆ tag followed by a tobacco etch virus cleavage sequence. The plasmid was transformed into E. coli strain BL21(DE3) Rosetta T1R for protein expression. In both constructs, all three native cysteines (Cys-40, Cys-43, and Cys-44) were mutated into alanines. In addition, two E_FL mutants previously described by Cohen et al. (43) were prepared by site-directed mutagenesis: (i) P54A (E_P54A) and (ii) V56A/Y57A/V58A/Y59A (E_4ALA).

Protein Expression and Purification

Non-labeled E protein was produced by growing the culture in Terrific broth medium at 37 °C until the culture density reached an A₆₀₀ of 2. Protein expression was induced by adding 0.5 mm isopropyl 1-thio-β-d-galactopyranoside and growing the culture overnight at 18 °C. The cells were harvested by centrifugation at 7,500 × g and stored at −80 °C.

Stable isotope-labeled E protein was produced by growing the culture in LB medium at 37 °C. When the culture density reached an A₆₀₀ of 0.7, the medium was exchanged to M9 minimal medium at 25% of the initial volume to achieve a high density culture, as described previously (50). The M9 medium was appropriately supplemented with ¹⁵N-NH₄Cl and [¹³C]glucose (Cambridge Isotope Laboratories) to produce ¹⁵N-labeled and ¹⁵N/¹³C-labeled protein. Cultures were further grown for 1 h before inducing protein expression with 0.5 mm isopropyl 1-thio-β-d-galactopyranoside at 18 °C. After 6 h, cells were harvested as described above and stored at −80 °C.

Frozen cell pellets were resuspended in lysis buffer (20 mm Tris, pH 8.0, 300 mm NaCl, 5 mm imidazole, 2 mm β-mercaptoethanol, and 10% glycerol) supplemented with 1 mm PMSF and 1.5% Triton X-100. The cells were completely lysed by sonication and microfluidization. Insoluble particles were removed by centrifugation at 40,000 × g, and the supernatant was applied onto a pre-equilibrated nickel-nitrilotriacetic acid resin (Bio-Rad Profinity IMAC Ni²⁺-charged). The resin was washed with 20 mm Tris, pH 8.0, 300 mm NaCl, 20 mm imidazole, 2 mm β-mercaptoethanol, and 10% glycerol. Bound peptide was eluted in 20 mm Tris, pH 8.0, 300 mm NaCl, 250 mm imidazole, 2 mm β-mercaptoethanol, 10% glycerol, and 5 mm myristyl sulfobetaine (C14SB). E_TR protein was directly TCA-precipitated and lyophilized, whereas E_FL was subjected to tobacco etch virus protease cleavage for 3 h at 30 °C prior to precipitation and lyophilization. Further purification was achieved by using reversed-phase HPLC on a Phenomenex Jupiter C4 semipreparative column (250 × 10 mm, 300-Å pore size, 5-μm particle size). Lyophilized peptide was dissolved with 1% TFA in acetonitrile and separated under an isopropyl alcohol-acetonitrile linear gradient (4:1 (v/v) with 0.1% TFA). The identity and purity of peptide fractions were confirmed by SDS-PAGE and MALDI-TOF MS.

Gel Electrophoresis

Standard SDS-PAGE was performed in 13.5% Tris-glycine gel with TGS running buffer and stained with Coomassie Blue G-250. SDS-NuPAGE was performed in 4–12% NuPAGE® BisTris gel (Invitrogen) with NuPAGE® MES SDS running buffer and stained with SimplyBlue^TM SafeStain (Invitrogen) according to the manufacturer's protocol.

To perform electrophoresis in the presence of perfluorooctanoic acid (PFO) detergent, we modified Invitrogen's SDS-NuPAGE protocol by replacing SDS with PFO. Lyophilized peptide was dissolved in sample buffer containing 4% PFO and heated at 65 °C for 5 min prior to loading. The gel was run at 80 V for 2–3 h with MES running buffer containing 0.5% PFO.

Blue native PAGE was performed as described previously (51). Lyophilized peptide was solubilized (0.1 mm) in sample buffer containing 25 mm SDS and either 25, 50, or 100 mm DPC. Aquaporin Z in 20 mm SDS (heated at 65 °C for 10 min) was included as an additional molecular weight marker.

Fourier Transform Infrared Spectroscopy

Sample preparation, data collection, and H/D exchange were performed essentially as described (32) on a Nicolet Nexus spectrometer (Madison, WI). The peptides were incorporated in multilamellar liposomes by dissolving a dry mixture of 1,2-dimyristoyl-sn-glycero-3-phosphate (DMPC; Avanti Polar Lipids) and lyophilized peptide in HFIP at a 50:1 molar ratio. Fourier self-deconvolution was performed for some spectra using the following parameters: full width at half height, 20 cm⁻¹; narrowing factor, k = 1.5) (52).

Analytical Ultracentrifugation

Sedimentation equilibrium experiments were performed using a Beckman XL-I analytical ultracentrifuge at 20 °C (53) and monitored by measuring the absorbance at 280 nm. Lyophilized E_TR peptides were dissolved at A₂₈₀ of 0.3, 0.5, and 0.8 (12-mm path length cell) in 20 mm sodium phosphate, pH 5.5; 50 mm NaCl; and for detergent either 5 mm C14SB, 100 mm DPC, or 12.5, 25, or 50 mm SDS. To match the density of the SDS-DPC mixture, D₂O was added at 61.6, 65, and 72.4%, respectively, to each SDS concentration. The samples were centrifuged in six-channel charcoal-filled Epon centerpieces using quartz windows. A radial distribution profile was acquired after sufficient time to reach equilibrium, as tested by HeteroAnalysis. The data were processed and fitted to several monomer/n-mer models in SEDFIT and SEDPHAT (54).

Circular Dichroism

CD data were acquired on a Chirascan CD spectrometer (Applied Photophysics) using a 0.2-mm quartz cuvette (Hellma). E_TR peptide samples were dissolved at 1 mg/ml in 20 mm sodium phosphate, 50 mm NaCl, pH 5.5, 100 mm DPC, with or without 50 mm SDS. CD spectra were acquired from 180 to 260 nm with a 1-nm spectral bandwidth and 3 replicates/spectra. Data points with excessive absorbance were excluded. After baseline subtraction, the data were analyzed in Dichroweb (55) by using the CDSSTR method (56) and the SMP180 reference set (57).

NMR Sample Preparation

Approximately 1.2 mg of lyophilized E_TR protein was solubilized in 100 μl of methanol and dried under a dry stream of N₂ gas, resulting in a thin protein film deposit. The tube was placed in a vacuum lyophilizer overnight to remove any residual methanol. The thin protein film was then solubilized with sample buffer containing 20 mm sodium phosphate, pH 5.5, 50 mm NaCl, and 50 mm SDS. The sample was vortexed and sonicated several times until a clear solution was obtained, indicating protein reconstitution into detergent micelles.

For paramagnetic relaxation enhancement (PRE) experiments, a single point mutation (S60C) was introduced into E_TR by site-directed mutagenesis using appropriate sets of primers. Expression and purification protocol of the E_TR-S60C mutant was the same as that of E_TR protein. For labeling, 0.3 mm ¹⁵N-labeled E_TR-S60C was dissolved in 20 mm sodium phosphate, 50 mm NaCl, 200 mm SDS, and 0.8 mm DTT at pH 5.5 and split into two equal portions for parallel labeling with (1-oxyl-2,2,5,5-tetramethyl-Δ³-pyrroline-3-methyl) methanethiosulfonate (MTSSL) (TorontoResearch Chemicals Inc.) and a diamagnetic analog of MTSSL: (1-acetyl-2,2,5,5-tetramethyl-Δ³-pyrroline-3-methyl) methanethiosulfonate (dMTSSL; Toronto Research Chemicals Inc.). A 10-fold molar excess of both reagents was added from 75 mm stocks in methanol. The sample was vortexed for 30 min at high speed and incubated overnight at room temperature. A centrifugal filter unit (10,000 molecular weight cut-off; Millipore Corp.) was used to remove excess of both reagents. Labeled samples were washed four times by concentrating to 100 μl. After a fourth wash, the sample was concentrated to 180 μl for NMR measurements.

Partial alignment of the E_TR protein-micelle complexes relative to magnetic field was obtained by using stretched polyacrylamide hydrogels (58, 59). A 7% polyacrylamide gel was polymerized in a gel chamber of 5.4-mm inner diameter. After complete polymerization, gels were washed in H₂O overnight and then twice with sample buffer containing 20 mm sodium phosphate and 50 mm NaCl at pH 5.5. The gels were then completely dried at room temperature. The protein solution containing E_TR/SDS was soaked into the dried gels over 2 days to ensure complete rehydration. The hydrated 7% gel was then compressed into a 4.2-mm inner diameter open-ended tube using the gel press assembly (New Era Enterprise, Inc.).

NMR Spectroscopy

NMR experiments were performed at 308 K using an Avance-II 700 NMR spectrometer with cryogenic probe. Sodium 2,2-dimethyl-2-silapentane-5-sulfonate was used as the internal reference for ¹H nuclei. The chemical shifts of ¹³C and ¹⁵N nuclei were calculated from the ¹H chemical shifts. The NMR data were processed using TopSpin version 3.1 and analyzed using CARA. Sequence-specific assignment of backbone ¹H^N, ¹⁵N, ¹³C′, and ¹³C^α was achieved by using two-dimensional ¹H-¹⁵N TROSY-HSQC, three-dimensional HNCO, HN(CA)CO, HNCA, HN(CO)CA, and HNCACB experiments on a ¹⁵N/¹³C-labeled E_TR protein. Side-chain resonances were assigned using three-dimensional ¹⁵N-resolved NOESY-HSQC (80-, 100-, and 150-ms mixing time), (H)CCH-TOCSY, and ¹³C-resolved NOESY-HSQC (120-ms mixing time). To identify membrane-embedded residues, the NMR sample was lyophilized overnight and reconstituted in 99% D₂O. Immediately after reconstitution, two-dimensional ¹H-¹⁵N TROSY-HSQC was collected. For paramagnetic probe measurements, ¹⁵N-HSQC spectra were recorded in the presence and absence of 1 mm dry 5-doxyl stearic acid (5-DSA) or 16-doxyl stearic acid (16-DSA). Axially symmetric alignment tensor coefficients (axiality and rhombicity) were calculated using MODULE (60). The PRE effect was measured using ¹⁵N HSQC spectra of the S60C mutant before spin labeling and after MTSSL and dMTSSL labeling. The titration experiments with HMA, Nsp3a, or SH(45–65) were performed with ¹⁵N-labeled E_TR in a 1:4 molar ratio of SDS/DPC micelles at 318 K. Chemical shift perturbation (CSP) values were calculated using the following formula.

Structure Calculation

NOE distance restraints were obtained from ¹⁵N NOESY-HSQC (mixing time 80, 100, and 150 ms) and ¹³C NOESY-HSQC (mixing time 120 ms) spectra, respectively. Backbone dihedral angle restraints (ϕ and ψ) were derived from ¹³C′, ¹³C^α, ¹³C^β, ¹H^α, and ¹H^β chemical shift values using TALOS+ (61). The short range and medium range NOE connectivities were used to establish the sequence-specific ¹H NMR assignment and to identify elements of the regular secondary structure. Hydrogen bonds were derived from the H/D exchange experiment and NOE connectivity.

Distance restraints were obtained from the measured PRE effect using the procedures described previously (62–64). The intensities of cross-peaks in the MTSSL (Ip) and dMTSSL (Id) were calculated in CARA. The correlation time was set to 10 ns. The ratios of intensities (Ip/Id) were normalized against a set of the eight highest Ip/Id ratios, which were assumed to belong to peaks unaffected by PRE. For peaks with ratios below 0.15, no lower distance restraints were used, whereas upper restraints were set to 15 Å. For peaks with ratios above 0.9, only upper restraints of 25 Å were utilized. For peaks with ratios between 0.15 and 0.9, upper and lower distance restraints were generated using ±3 Å margins.

Structure calculations were performed using CYANA version 3.0 (65, 66) and visualized using PyMOL (Schroedinger LLC, New York). CNS 1.3 (67, 68) was used to refine the structure using the standard simulated annealing protocol. All of the restraints used in the calculations to obtain a total of 15 structures and all of the structure statistics are summarized in Table 1.

TABLE 1.

Restraints and structure statistics for the selected 15 structures of E_TR

NMR restraints
Total unambiguous distance restraints	2,258
Intraresidual	1,334
Sequential (|i − j| = 1)	230
Short-range (|i − j| ≤ 1)	1,564
Medium (2 ≤ |i − j| ≤ 4)	321
Long range (|i − j| ≥ 5)	11
Dihedral angle restraints	85
Hydrogen bond restraintsa	20
RDC restraints	44
PRE restraints	38
Root mean square deviation from the experimental residual dipolar couplings (Hz)
1DNH	0.71 ± 0.03
Root mean square deviation from the average atomic coordinates (residues 12–63, Å)b
Backbone atoms	0.27 ± 0.11
All heavy atoms	0.70 ± 0.13
Ramachandran analysis (%)
Residues in most favored regions	87.5
Residues in additional allowed regions	12.5
Residues in generously allowed regions	0.0
Residues in disallowed regions	0.0

^a Backbone hydrogen bonds of α-helix were applied to regions confirmed to be α-helical, according to the local NOE pattern and H^N-H₂O chemical exchange experiments.

^b Statistics were calculated and averaged over an ensemble of 15 structures with lowest target function according to CYANA.

Single Channel Activity Measurement

Ion channel activity of E_TR was measured by using Nanion Port-a-Patch®. Briefly, giant unilamellar vesicles of 1,2-diphytanoyl-sn-glycero-3-phosphocholine containing 10% cholesterol were prepared in 1 m sorbitol using Nanion Vesicle Prep Pro®, following the manufacturer's protocol. Into 100 μl of a giant unilamellar vesicle solution, 0.5 μl of E_TR peptide solution in ethanol (0.1 mg/ml) was added and incubated for 1 h at room temperature. E_TR-containing giant unilamellar vesicles were subsequently deposited onto 6–8 megaohm NPC®-1 chips (Nanion). Conductance was measured under symmetrical buffer conditions (10 mm HEPES, 500 mm NaCl, pH 5.5).

Surface Plasmon Resonance

The nsp3a sequence was subcloned from pcDNA3(+) into pET28b upstream of a C-terminal His₆ tag for expression in E. coli. The protein was expressed and purified as described previously by Serrano et al. (69). A negative control, consisting of C-terminal peptide from the small hydrophobic (SH) protein of human respiratory syncytial virus (RSV SH(45–65)), was synthesized by standard solid phase and purified by reverse-phase HPLC. Surface plasmon resonance measurements were performed on a Biacore 3000 system (GE Healthcare) using 10 mm phosphate buffer at pH 6.5, 100 mm NaCl, 3 mm EDTA, 0.05% n-octyl-β-d-glucopyranoside, and 0.27% C14-betaine at 25 °C. E_TR was immobilized to 15,000 RU onto a research grade CM5 sensor chip (GE Healthcare) using standard amine-coupling chemistry. Briefly, a buffer-equilibrated carboxymethyl dextran surface was activated with a 10-min injection of a 1:1 mixture of 0.05 m N-hydroxysuccinimide and 0.2 m N-ethyl-N-[3-(diethylamino)propyl]carbodiimide. E_TR peptides dissolved in 10 mm sodium acetate, 15 mm DPC (pH 5.0) were passed over the activated surface to achieve the desired response level. Another 10-min injection of 1 m ethanolamine-HCl (pH 8.5) was used to deactivate the surface and remove any non-covalently bound protein. Kinetic measurements of immobilized E_TR association with Nsp3a and SH(45–65) (49 nm to 25 μm in 10 2-fold serial dilutions) were performed with a 1-min association phase and 5-min dissociation phase at a 30 μl/min flow rate. Each concentration was tested in duplicate. No regeneration was necessary because all complexes dissociated within the monitored time. Sensorgrams were double-referenced (70) and globally fit to a steady-state model to obtain affinity values.

RESULTS AND DISCUSSION

Expression and Purification of SARS-CoV E Protein

Initially, we successfully expressed and purified full-length SARS-CoV E protein (E_FL, Fig. 1B) by using either a β-barrel (71) or MBP as fusion tags. However, the yield of pure protein was low due to the presence of truncations (not shown). Nevertheless, the pure sample obtained (Fig. 1, C and D) was sufficient for backbone assignment in SDS micelles, although not to produce a three-dimensional model. Therefore, a series of hexahistidine-tagged SARS-CoV E constructs were screened to obtain an expressing and well behaved sample. The best construct, encompassing residues 8–65, was successfully expressed in E. coli and purified by affinity chromatography in milligram amounts without any enzymatic cleavage steps. This truncated construct (E_TR; see Fig. 1B) has an N-terminal His tag and a 16-residue linker that connects it to residues 8–65. The purified peptide appeared in MALDI-TOF MS as a single-charged peak at 8,997 Da and a double-charged peak at 4,512 Da, consistent with the calculated molecular mass of E_TR, 8,995 Da, and a small proportion of larger (dimer to pentamer) oligomers (Fig. 1E). After HPLC, the E_TR monomer (9 kDa) showed anomalous migration in standard SDS-PAGE (Fig. 1F), as reported previously for the full-length protein (31).

Identification of the Membrane-embedded Region of E_TR by NMR

Screening of reconstitution conditions identified SDS as the best environment to achieve good peak dispersion in both dimensions (Fig. 2A). Comparison of ¹H-¹⁵N TROSY-HSQC spectra in water and in 99% D₂O (Fig. 2, A and B) identified a stretch of 21 residues, from Leu-18 to Leu-39, protected from H/D exchange. The same residues were protected from H/D exchange in E_FL (not shown), which indicates that both E_TR and E_FL have only one TM domain.

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.

The topology of E_TR was also delineated by introducing the paramagnetic probes 5-DSA and 16-DSA. Upon the addition of 5-DSA, a slight intensity reduction was observed around residues 11–20 and 40–55 (Fig. 2C), suggesting that these residues are located at or near the micelle surface. The addition of 16-DSA caused pronounced intensity reduction in the stretch of TM residues 19–40 and periodically in the stretch 48–61 (Fig. 2D). Together with the H/D exchange data (Fig. 2, A and B), these results again demonstrate the presence of a single TM domain and suggest that the stretch of residues 55–65 corresponds to a domain bound to the surface of the micelle. Finally, a ¹H-¹⁵N steady-state heteronuclear NOE experiment (Fig. 2E) showed that the protein forms a well folded structure, with high ¹H/¹⁵N NOE values. However, more dynamic regions (lower ¹H/¹⁵N NOE values) can be observed (i) at the N terminus and (ii) at residues 46–55, which connects the TM domain to the proposed membrane-bound domain. The latter is predicted to be α-helical (Fig. 2F).

NMR Structure Determination of E_TR in SDS Micelles; Structure Calculation

The restraints shown in Table 1 were used in a calculation to obtain a total of 15 structures, with a root mean square deviation of 0.27 ± 0.11 and 0.70 ± 0.13 Å for backbone and all heavy atoms, respectively (Fig. 3A). A longer α-helix (residues 15–45) encompasses the TM domain, which is connected to another shorter C-terminal α-helix (residues 55–65) by a flexible (see Fig. 2E) linker domain (residues 46–54), forming an L-shape. The short extramembrane helix may be partially bound to the micelle surface, as suggested by the pattern of intensity attenuation of paramagnetic reagents (see Fig. 2, B and C). The most affected residues, which would face the micelle surface, are Val-52, Thr-55, Tyr-59, and Lys-63 (Fig. 3B).

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.

The TM α-helix has a slight bend at residues 26–30, consistent with previous results obtained for the synthetic TM domain in DPC micelles (34). A kink near this location is suggested by the short distance (2.0 ± 0.1 Å) between Thr-30 Hγ and the carbonyl oxygen at Phe-26, in the range of a hydrogen bond (Fig. 3C).

Effect of Truncation and Environment on E_TR Secondary Structure

To assess the effect of the truncation and the presence of a His tag on the E_TR secondary structure, E_TR and E_FL were compared. The possible effect of the reconstitution environment was also determined.

CD and IR Spectra of E_TR and E_FL in Detergent and Lipid Membranes

The CD spectra of E_TR in DPC, SDS, and mixed (1:2 molar ratio) SDS/DPC micelles are almost superimposable (Fig. 4A) with minima at 209 and 222 nm. Also, these data are entirely consistent with the CD spectra of E_FL, which was predominantly α-helical in both SDS and DPC micelles (71). When reconstituted in DMPC membranes, E_TR and E_FL produced an almost identical spectrum, with an amide I band centered at 1,655 cm⁻¹ (Fig. 4B) characteristic of a predominantly α-helical conformation. Overall, these data show that both E_TR and E_FL (i) are predominantly α-helical and (ii) have a secondary structure that is not significantly affected by the reconstitution environment, supporting the relevance of the E_TR structure (Fig. 3).

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.

¹³Cα Chemical Shifts

¹³Cα chemical shifts are highly correlated with secondary structure (72, 73). Comparison of ¹³Cα chemical shifts of E_TR and E_FL in SDS (Fig. 4C) shows that E_TR shifts (red) are almost identical to their counterpart residues (residues 8–65) in E_FL (blue). In E_FL, the shifts for the last 10 residues (residues 66–76) are predicted to correspond to random coil, which is consistent with results obtained previously with a synthetic peptide spanning residues 59–76 (39). This peptide produced a broad amide I band in the IR spectrum centered around 1,650 cm⁻¹ and immediately experienced complete H/D exchange.

When the ¹³C shifts for E_TR were compared for SDS and an SDS/DPC mixture (1:4 molar ratio), only the N-terminal region (residues 7–15) showed minor differences. Finally, a comparison between E_FL and E_TR in an SDS/DPC mixture (1:4 molar ratio) also revealed an almost identical pattern in the C-terminal tail (not shown), although the spectral resolution for E_FL was reduced in other regions. Overall, these results are consistent with those shown above (Fig. 4, A and B), indicating that the detergent used, the tag, and the truncation has a minimal on E_TR secondary structure.

Ion Channel Activity of E_TR and Its Inhibition by HMA

Purified E_FL has channel activity (71), which is inhibited by the drug HMA (30). An I/V plot obtained in a symmetrical 0.5 m NaCl experiment for purified E_TR in 1,2-diphytanoyl-sn-glycero-3-phosphocholine (Fig. 5A) was used to determine a conductance of 0.39 ± 0.02 nanosiemens. For comparison, synthetic full-length SARS-CoV E and E_TM (residues 7–38) produced single channel conductances of 0.19 ± 0.06 picosiemens and 0.18 ± 0.12 nanosiemens in 1 m NaCl (35), although the values were higher in 1 m KCl, with 0.37 ± 0.16 and 0.31 ± 0.12 for full-length SARS-CoV E and E_TM, respectively. The lower conductance observed in synthetic samples may be due to extraneous modifications or impurities resulting from exposure to harsh chemicals. Representative traces of E_TR channel activity (Fig. 5B) and complete inhibition after the addition of 10 μm HMA (Fig. 5C) suggest that E_TR is entirely functional.

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.

To determine the binding site for HMA, we measured the differences in CSP values before and after the addition of the drug (Fig. 5, D and E). In SDS, the average CSP value was low, 0.006 ppm (Fig. 5F), even at an HMA/E_TR 10:1 molar ratio, suggesting no significant binding. However, the same panel shows that the addition of HMA to E_TR in mixed SDS/DPC (1:4 molar ratio) micelles produced an average CSP value of 0.013 ppm, even at an HMA/E_TR 2:1 molar ratio.

Except for Asn-64 and Leu-65, the residues that showed significant CSP (CSP ≥ 0.025 ppm) clustered near the membrane interface regions of the TM domain (Fig. 5F, see arrows). Near the N-terminal side of the TM domain, the most affected were Glu-8, Gly-10, Thr-11, Val-14, Asn-15, and Ser-16, the latter two consistent with observations made on the TM channel in DPC (34). At the C-terminal end of the TM, Leu-37 was the most affected, suggesting that the interaction of HMA at Hϵ of Arg-38 reported previously (34) may have been an artifact due to the use of a TM peptide.

Oligomeric State of E_TR

Gel Electrophoresis

The localized changes in chemical shifts observed after HMA addition to E_TR in mixed DPC/SDS micelles (Fig. 5E), but not in SDS micelles (Fig. 5D), suggest that binding sites for HMA may have been induced after DPC addition to SDS, possibly through E_TR increased oligomerization and population of the pentameric form. Thus, we examined the oligomeric state of E_TR using gel electrophoresis performed in the presence of SDS or an SDS/DPC mixture. In contrast with the results obtained in normal SDS-PAGE (Fig. 1), we used NuPAGE, where E_TR migrated with its expected monomer molecular weight, as a single band at 9 kDa (Fig. 6A). No oligomerization is consistent with the lack of interaction between HMA and E_TR observed in SDS micelles.

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.

To test the effect of DPC, in a blue native polyacrylamide gel, a constant SDS concentration (25 mm) was titrated with increasing DPC (Fig. 6B), from 1:1 to a 1:4 molar ratio. Bands consistent with dimers and trimers were observed at 1:1 and 1:2 molar ratios, whereas tetramers and pentamers were observed at a 1:4 molar ratio. This increasing oligomerization is again consistent with the binding of HMA observed in the presence of DPC.

Sedimentation Equilibrium, SDS/DPC Micelles

For a sample corresponding to a 1:4 molar ratio of SDS/DPC, the equilibrium sedimentation data (Fig. 7A) could be fitted to several oligomeric models, from trimers to heptamers (Fig. 7B). Similar ambiguous results were obtained for a 1:2 molar ratio SDS/DPC mixture (Fig. 7C), suggesting that E_TR forms a mixture of oligomers in SDS/DPC micelles, consistent with electrophoresis results (Fig. 6). The fit of the traces (Fig. 7A) to a monomer-pentamer model produced an apparent K_a of 1.25 × 10¹⁵ m⁻⁴ (i.e. 6 × 10³ m⁻¹) or a K_d for monomer-monomer interaction of 0.17 mm and a molar fraction standard free energy change (74), ΔG_x⁰, of approximately −4 kcal·mol⁻¹. Overall, the above results show that higher oligomeric states, including pentamers, are observed when increasing the DPC concentration, consistent with the larger shifts observed after adding HMA to mixed DPC/SDS micelles.

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.

E_TR Forms Pentamers in C14 Betaine and PFO

We have shown previously that SARS-CoV E TM domain and E_FL form pentamers (36, 71). When E_TR was solubilized in C14 betaine detergent (Fig. 7D), data could be optimally fitted to a monomer-pentamer model (K_a = 10¹⁶ m⁻⁴) with a significantly lower χ² than that obtained in SDS/DPC. Also, the mobility of E_TR in PFO-NuPAGE (Fig. 7E) corresponds to a molecular mass of 45 kDa (i.e. a pentameric form). Thus, E-TM, E_TR, and E_FL show a similar oligomerization behavior, being able to form pentameric channels that are inhibited by HMA.

Interaction of E_TR with Nsp3a

The interaction of Nsp3 with SARS-CoV E protein, thought to be related to E ubiquitination, was previously mapped to an N-terminal acidic domain, Nsp3a (49). Although the site of interaction with E is not known, it is likely to involve the C-terminal tail because it is the largest extramembrane domain. Thus, to test that the conformation of E_TR is structurally equivalent to the corresponding sequence in E_FL, we tested its ability to interact with Nsp3a (49) using surface plasmon resonance.

As a negative control, the mock peptide RSV SH(45–65) (see “Materials and Methods”) was used to bind to immobilized E_TR in detergent (see “Materials and Methods”) (Fig. 8A). Binding and unbinding of RSV SH(45–65) was very slow (>1 min), and the data could not be fitted to any model, suggesting a nonspecific interaction.

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).

In contrast, rapid and reversible interaction was observed using purified Nsp3a (Fig. 8B). These data could be fitted with a stoichiometry of 1:1, although the end point could only be estimated. From that model, affinity was determined as 1.6 mm, although we note that binding is already evident at concentrations as small as 1–10 μm (see Fig. 8B, inset).

The residues involved in the interaction between Nsp3a and E_TR were identified by the differences in CSP values before and after the addition of Nsp3a to E_TR in SDS/DPC micelles (1:4 molar ratio). Large chemical shift changes at Leu-39, Val-49, and Leu-65 (Fig. 8C) indicated a potential binding site at the C-terminal tail, whereas the central TM region did not experience any change. Overall, these data suggest that E_TR has a structure in SDS/DPC micelles that is similar to the native fold of E_FL in biological membranes.

Structural Discordance at the C-terminal Tail

Having determined the suitability of the construct used by comparison with the full-length protein and shown that the nature of the detergent did not affect the secondary structure significantly, we investigated the structure of the C-terminal extramembrane domain of E_TR and its apparent contrast with secondary structure predictions.

The secondary structure predictions clearly suggest the presence of a β-turn-β motif in the C-terminal region, not only in SARS-CoV E but also in other representative sequences of other coronaviruses (see Fig. 1A). Indeed, we have reported previously that the synthetic peptide E_46–60, which encompasses this putative β-hairpin, folds as β-strands, is completely resistant to H/D exchange, and has a very high tendency to aggregate in solution (39). However, we have shown here that in the context of E_FL or E_TR, this domain does not adopt a β-structure. To test if at least some part of the population adopts this structure in lipid membranes, we mutated E_FL residues 56–59 to increase the predicted helicity of this part of the molecule (43), from β-branched Val or bulky Tyr to small side chain Ala, to obtain the construct E_4ALA. Last, to test the effect of the conserved Pro-54 at the center of this putative motif, we generated the mutant E_P54A.

The IR spectrum of these mutants when reconstituted in DMPC membranes (Fig. 9A) shows a similar amide I band. However, for the mutant E_4ALA, two shoulders are eliminated (see arrows). This indicates that these shoulders in E_FL do not represent misfolded protein, but they may correspond to the bona fide β-structure conformation present in a small part of the population. However, the significance of these mutations is not completely clear because, although the mutations introduced in E_4ALA prevented Golgi complex accumulation when the C terminus tail of SARS-E was coupled to VSV-G (43), a similar effect was also observed for the P54A mutant, which in our IR spectrum showed no obvious differences with respect to E_FL.

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.

As stated above, the synthetic peptide E_46–60 (39), which encompasses the predicted β-hairpin in SARS-CoV E, was found to produce ∼100% β-structure and was completely resistant to H/D exchange (Fig. 9C). Combined with the effect observed for the E_4ALA mutant, we propose that this β structure may be in dynamic equilibrium with the much more abundant α-helical form (Fig. 9D). A delicate balance between these two forms may alter processes in the infected cell (e.g. membrane scission, binding to protein partners, or E protein localization).

Finally, the HMA titration results showed large CSP values for residues Val-49 and Leu-65, which are far apart in the sequence. Using the SymmDock server (75, 76), a reconstructed E_TR pentameric model was obtained based on the published structure of the E-TM pentamer (34) and the current E_TR structure. The model suggests that these two residues may be in fact spatially close (Fig. 10) and belong to different monomers, providing a rationale for the observation above.

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.

CoV E proteins have been proposed to have at least two roles. One is related to their TM channel domain. This would be active in the secretory pathway, altering lumenal environments and rearranging secretory organelles and leading to efficient trafficking of virions (38, 77). The other would be related to their extramembrane domains, particularly the C-terminal domain. This is involved in protein-protein interactions and targeting, among other roles.

E protein participates in M-M and E-M interactions (17, 44), which are interesting targets for drug discovery. Also, formation of viral particles appears to be facilitated by a broad range of E sequences (78), which suggests that a common topology is more important than sequence requirements. E proteins have been suggested to act as chaperones during packaging (79), but the precise mechanism by which this takes place is not known. In this context, the structure determined here sheds light on a critical domain present in most CoV E proteins.