Solution structure of the severe acute respiratory syndrome-coronavirus heptad repeat 2 domain in the prefusion state

Abstract

The envelope glycoprotein, termed the spike protein, of severe acute respiratory syndrome coronavirus (SARS-CoV) is known to mediate viral entry. Similar to other class 1 viral fusion proteins, the heptad repeat regions of SARS-CoV spike are thought to undergo conformational changes from a prefusion form to a subsequent post-fusion form that enables fusion of the viral and host membranes. Recently, the structure of a post-fusion form of SARS-CoV spike, which consists of isolated domains of heptad repeats 1 and 2 (HR1 and HR2), has been determined by x-ray crystallography. To date there is no structural information for the prefusion conformations of SARS-CoV HR1 and HR2. In this work we present the NMR structure of the HR2 domain (residues 1141-1193) from SARS-CoV (termed S2-HR2) in the presence of the co-solvent trifluoroethanol. We find that in the absence of HR1, S2-HR2 forms a coiled coil symmetric trimer with a complex molecular mass of 18 kDa. The S2-HR2 structure, which is the first example of the prefusion form of coronavirus envelope, supports the current model of viral membrane fusion and gives insight into the design of structure-based antagonists of SARS.

FIGURE 1

¹⁵N-edited HSQC of SARS-CoV S2-HR2. The sample conditions were 1 mm S2-HR2 monomer, 10 mm NaPO₄, pH 7.0, 30% TFE-d3 at 25 °C. Horizontal lines connect the side chain amide proton pairs of asparagine and glutamine residues.

FIGURE 2

Secondary structure of SARS-CoV S2-HR2.a, secondary chemical shift of S2-HR2 ¹³C_α (gray) and ¹³C′ (white) with respect to random coil values. Random coil values were taken from Wishart and Case (64). b, HNOE of S2-HR2.

FIGURE 3

CLEANEX-PM ¹⁵N-edited HSQC. The sample conditions were 1 mm of S2-HR2 monomer, 10 mm NaPO₄, pH 7.0, 30% TFE-d3 at 25 °C with a mixing time of 100 ms. Note that residues Thr⁴ and Ser⁵, which occur in an unstructured region at the N terminus, exhibit negative contours, presumably because of ROE effects and/or intermolecular NOEs with H₂O (49). The absence of a correlations for residues 17-47 suggests that they are involved in H-bonds. The absence of correlations for residues 14-16 and 50 may suggest the presence of H-bonds. The other missing correlations include those of residues 1-3, which are also missing from the ¹⁵N-edited HSQC (residue 6 is a proline).

FIGURE 4

Intermolecular NOEs of SARS-CoV S2-HR2 showing intersubunit contacts involving the helix. Selected strips from the three-dimensional F1-filtered F2-edited ¹H-¹³C NOESY-HSQC spectrum (mixing time of 120 ms) recorded on a 1:1 mixture of ¹²C/¹⁴N-and ¹³C/¹⁵N-labled S2-HR2.

FIGURE 5

Solution structure of SARS-CoV S2-HR2.a, ensemble of 30 low energy structures of showing the superimposition of the backbone atoms. b, ribbon representation of the minimized mean structure. c, electrostatic map of the minimized mean structure. In a and b, subunits A, B, and C are colored red, green, and blue, respectively. The direction of the viral membrane is shown by an arrow.

FIGURE 6

Structural features of SARS-CoV S2-HR2.a, intermolecular contacts between the S2-HR helices. b, helical wheel representation of S2-HR2 residues Asn¹⁷-Leu⁴⁷ looking down the helical axis, starting at the N-terminal end. There are seven residues/heptad where the individual positions of the seven residues are denoted by the letters a-f. The residues in the a and d positions make up the hydrophobic interface of the trimeric coiled coil of the S2-HR2 structure. The a positions are highlighted purple, and the d positions are highlighted green.