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. 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 has been found to be a pentamer, with a hydrophobic pore that appears to function as an ion channel. We identified only a 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.

Figure 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 (Figure S1). 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.

Figure 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 ${\mathrm{\sigma }}_{11}=54.5\mathrm{p}\mathrm{p}\mathrm{m},{\mathrm{\sigma }}_{22}=82.8\mathrm{p}\mathrm{p}\mathrm{m},{\mathrm{\sigma }}_{33}=227.1\mathrm{p}\mathrm{p}\mathrm{m}$. The amino acid sequence for E_12–37 with its N-terminal SNA tag is: SNAL₁₂IVNSVLLF₂₀LAFVVFLLVT₃₀LAILTAL₃₇.

Figure 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 Ala11, 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, isotopic labeling included the Ala residue (denoted as Ala11) from the TEV cleavage site, and the calculated PISA wheel used a somewhat larger ${\mathrm{\sigma }}_{33}$ of 232.1 ppm.

Figure 4.

¹³C-¹³C correlation spectra of a 50:50 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.

Figure 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.

Figure 6.

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