Dimeric Transmembrane Structure of the SARS-CoV-2 E Protein

Abstract

The SARS-CoV-2 E protein is a transmembrane (TM) protein with its N-terminus exposed on the external surface of the virus. At debate is its oligomeric state, let alone its function. Here, the TM structure of the E protein is characterized by oriented sample and magic angle spinning solid-state NMR in lipid bilayers and refined by molecular dynamics simulations. This protein was previously found to be a pentamer, with a hydrophobic pore that appears to function as an ion channel. We identify only a front-to-front, symmetric helix-helix interface, leading to a dimeric structure that does not support channel activity. The two helices have a tilt angle of only 6°, resulting in an extended interface dominated by Leu and Val sidechains. While residues Val14-Thr35 are almost all buried in the hydrophobic region of the membrane, Asn15 lines a water-filled pocket that potentially serves as a drug-binding site. The E and other viral proteins may adopt different oligomeric states to help perform multiple functions.

Fig. 1. SDS-PAGE gels of purified E constructs.

a E_12–65. b E_12–37. c E_1–52. d E_7–43. e E_8–41. All of these constructs included leading Ser-Asn-Ala residues from the TEV cleavage site (Supplementary Fig. 1). The molecular weight of each construct is shown below its designation. The observed bands for monomer and dimer next to lane 2 in each gel are indicated. All display dominant bands for the dimer.

Fig. 2. ssNMR spectra of uniformly ¹⁵N labeled E_12–37 in aligned POPC/POPG bilayers.

a 1D ¹⁵N spectrum. b 2D PISEMA spectrum of the same sample, with contours down to the spectral level as shown by the arrows on the 1D spectrum. Superimposed is a PISA wheel for a helix tilt angle of 6° with respect to the bilayer normal, calculated using a chemical shift tensor with σ₁₁ = 54.5 ppm, σ₂₂ = 82.8 ppm, σ₃₃ = 227.1 ppm and a dipolar coupling constant of 10.4 kHz. The amino acid sequence for E_12–37 with its N-terminal SNA tag is: SNAL₁₂IVNSVLLF₂₀LAFVVFLLVT₃₀LAILTAL₃₇.

Fig. 3. PISEMA spectra of ¹⁵N labeled E_12–37 peptides in POPC/POPG bilayers.

a ¹⁵N-labeling at Ile13 and Ile33. b ¹⁵N-labeling at Phe20, Phe23, and Phe26. c ¹⁵N-labeling at Ala22, Ala32, and Ala36. d ¹⁵N-labeling at Thr30 and Thr35. e ¹⁵N-labeling at Val14, Val17, Val24, Val25, and Val29. f ¹⁵N-labeling at Leu12, Leu18, Leu19, Leu21, Leu27, Leu28, Leu31, Leu34, and Leu37. Superimposed on the spectra is a PISA wheel calculated at a tilt of 6°. For the Ala spectrum, the calculated PISA wheel used a somewhat larger σ₃₃ of 232.1 ppm.

Fig. 4. ³C-¹³C correlation spectra of a 1:1 mixture of ¹³C-Leu labeled E_12–37 and ¹³C-Val labeled E_12–37 in POPC/POPG liposomes.

Data obtained at mixing times of (a) 600 ms and (b) 300 ms. Interhelical cross peaks are assigned. In (a), the signal to noise (S/N) ratios for ¹³C-Leu and ¹³C-Val cross peaks are: LCγ-VCα, 5.7; LCβ-VCα, 2.8; and LCα-VCα, 2.5. Interhelical cross peaks with S/N ratios above 2 are usually considered strong.

Fig. 5

¹³C-¹³C correlation spectrum of an equimolar mixture of ¹³C-Leu labeled E_12–37 and ¹³C-Phe labeled E_12–37 in POPC/POPG liposomes at a mixing time of 600 ms.

Fig. 6

Refinement of the E_12–37 dimer by restrained molecular dynamics simulations in POPC/POPG membranes.