Central ions and lateral asparagine/glutamine zippers stabilize the post-fusion hairpin conformation of the SARS coronavirus spike glycoprotein

Abstract

The coronavirus spike glycoprotein is a class I membrane fusion protein with two characteristic heptad repeat regions (HR1 and HR2) in its ectodomain. Here, we report the X-ray structure of a previously characterized HR1/HR2 complex of the severe acute respiratory syndrome coronavirus spike protein. As expected, the HR1 and HR2 segments are organized in antiparallel orientations within a rod-like molecule. The HR1 helices form an exceptionally long (120 A) internal coiled coil stabilized by hydrophobic and polar interactions. A striking arrangement of conserved asparagine and glutamine residues of HR1 propagates from two central chloride ions, providing hydrogen-bonding "zippers" that strongly constrain the path of the HR2 main chain, forcing it to adopt an extended conformation at either end of a short HR2 alpha-helix.

Fig. 1

The HR1 and HR2 regions in the coronavirus S glycoprotein. (A) Linear diagram indicating the relative locations of the segments described in the text. The S1 and S2 regions are labeled. FP: putative fusion peptide region. TM: transmembrane region. SP: signal peptide. (B) Sequence alignments of the HR1 and HR2 regions of coronavirus spike proteins of the three different groups (G1 to G3) and the unclassified SARS coronavirus (blue arrow) using ClustalW (Thompson et al., 1994). See the Supplementary material for the complete names of the viruses used. The numbers at the top line correspond to the SARS amino acid sequence, the structure of which is described in the text. The alignment is color-coded according to sequence conservation: red, strictly conserved; green, highly conserved; blue, conserved; black, variable. The alignment in the HR2N region was manually modified to match the structural superposition with the corresponding region of the MHV protein (PDB accession numbers 1WDF and 1WDG, Xu et al., 2004c). Residues with ordered electron density have a grey background in the SARS line. The cloned fragment contained all residues between the boxed columns (further highlighted with a vertical empty arrow below the sequence). Two additional lines at the bottom summarize the following: the “Register” line provides the abcdefg heptad repeat assignment with letters in black for the residues actually observed in a helical conformation in the structure, with the two HR1 stutters in grey background. Note the insertion of exactly two heptad repeats, both in HR1 and HR2, in the S protein of group 1 coronaviruses. The “Interaction” line shows the two salt bridges (1 and 2, see Fig. 2B) in a yellow background. In the case of HR1, this line provides also the residues participating in the asparagine/glutamine zipper shown in Fig. 3 (labeled “N”), the knob-into-hole interactions with either partner within the trimer (labeled B or C), and the residues lining the central cavity shown in Fig. 2B (labeled with a star). Residues forming salt bridges 1 and 2 connecting HR1 with HR2 have a yellow background, with a number below to indicate the partner in each chain. Note that salt bridge 1 is conserved, but is sometimes made with HR1 residue 900 (from the previous helical turn) instead of 903. The structure shows that the side chain of residue 900 can contact the HR2 1188 side chain equally well. Blue background columns indicate HR1 residues interacting with the two central chloride ions. Vertical grey background columns identify HR2 residues in the extended segments (HR2N and HR2C) that pack their side chains into hydrophobic pockets in the HR1 interhelical grooves.

Fig. 2

Overall structure: determinants of the hydrophobic core formation and central ions interactions. (A and B) Ribbon diagrams colored light and dark grey for the HR1 and HR2 polypeptides, respectively. Pink spheres on the central 3-fold axis indicate the chloride ions. In A, the axes of the HR1 helices in the trimer are drawn as green tubes, highlighting the two stutters in red. The coiled coil axis is dark red (vertical at the trimer center). The helical turns are numbered from N- to C-terminus for one of the subunits (black and white numbers are used for HR1 and HR2, respectively). The N- and C-terminal ends of the model are indicated for one HR1/HR2 heterodimer. The 2 columns between the vertical scale bar on the left and the ribbon diagram indicate the residues and the 3- and 4-residue repeat pattern of the side chains facing the 3-fold axis of the coiled coil. Black and red fonts indicate polar and non-polar side chains, respectively. Residues within green boxes are strictly conserved. Red boxes in the second column highlight the stutters. In B, the side chains of polar residues within the hydrophobic core are drawn in green and labeled. Water molecules are indicated as small red spheres. Inter chain salt bridges (1 and 2, labeled in blue, corresponding to Lys 903 to Glu 1188 and Lys 929 to Glu 1163, respectively) are also indicated (basic side chains are in blue, acidic in red). The central cavity is displayed as a gold surface. (C) Slab of the model viewed down the 3-fold axis to show the chloride ions. Pink arrows indicate the center of the slab in panel B: top and bottom panels display chloride ions 1 and 2, respectively. The hydrogen bonding network propagating from the central ions toward the outside—which highly constrains the HR2 main chain, is indicated. Several of the Asn and Gln residues labeled are part of the Asn/Gln zipper (see text). As a guide for orientation, the axes of the 3 HR1 α-helices are drawn and labeled in green. The top panel is a view from below the atom, and the bottom panel from above it, relative to panel 2B. (D) Helical wheel after correction for the stutters (as in the register line of Fig. 1B). HR1 left, HR2 middle: as in panel A, polar and non-polar side chains are black and red, respectively (notice the strong amphipathic character of the two helices). Positions a and d are highlighted within a circle with a red background. The right panel shows a diagram of the interactions in the 6-helix bundle.

Fig. 3

HR1/HR2 interactions: Asn/Gln zippers and hydrophobic pockets along the HR1 grooves. Left panel. Ribbon representation, colored and oriented as in Figs. 2A and B, in which all the main chain atoms of the HR2N and HR2C extended segments of one of the subunits are represented as ball and stick, as well as the side chains of the Asn and Gln residues participating in the zipper. The atoms are colored according to atom type: grey are carbon atoms (light and dark for atoms of HR1 and HR2, respectively), red and blue indicate oxygen and nitrogen atoms, respectively. Hydrogen bonds between Gln and Asn side chains and main chain atoms are displayed as hatched cyan tubes. At the top, the arrows indicate the segments that connect to the fusion peptide (FP) and trans-membrane (TM) region. The boxes indicate the regions blown up in the right panels. The α-helical 5 turns of HR2 are numbered. Right panels. The top and bottom panel zoom into the Asn/Gln zippers that constrain the HR2C and HR2N segments, respectively. The model was slightly rotated in each of the two panels, with respect to the view in the left panel, for clarity. Hydrophobic HR2 side chains fitting into pockets in the HR1 grooves are shown in green. Asn and Gln side chains are colored as in the left panel. All residues indicated are labeled, red boxes highlighting highly conserved residues. The interactions displayed in this figure, together with the interactions that form an N- and C-cap to the HR2 helix as described in the text, highly constrain the HR2N and HR2C main chain.

Fig. 4

Recapitulation of the current structural data on the fusion core of the SARS CoV glycoprotein S. The left panel (framed in red) displays the structure described in this report in a ribbons representation in which the three HR1 segments are in primary colors and HR2 in grey. The other three panels are labeled with the corresponding PDB accession code of the structure depicted (pdb codes 1BEQ and 1BEZ from Supekar et al., 2004, and 1WNC from Xu et al., 2004b). In all panels, both the trimeric molecule (top) and one subunit (bottom) are displayed. All panels are colored identically, with segments containing amino acids that are not present in our current model (in the left panel) colored white. The images are all at the same scale, with the horizontal bars providing a means to align them so that the N-terminus of the HR2 helix is at the same height in each panel. The bottom panel indicates the number of the N- and C-terminal ends of the constructs represented. At the top, a roughly-to-scale diagram of a putative “fused membrane”, with its aliphatic portion in blue and the hydrophilic lipid heads in orange, is drawn at about the expected distance from the structures, as deduced from the amino acids that are missing between the N- and C-termini and the membrane interacting segments of the protein, the N-terminal fusion peptide and the C-terminal trans-membrane region.

Supplementary Fig. 1

Mechanism of stutter formation. Examination of crick's angles along the helix (A) shows a progressive decrease of the values at each position. This reflects the regular 3.64 residues per turn observed in the helix and constant phase yield between successive residues of 98.9° rather than 3.5 and 102.8° expected for a perfect coiled coil. Thus, to keep in phase hydrophobic residues, a stutter must be inserted which cause a shift of around 50° in all values. At each stutter position (arrow), an increase of the local coiled coil radius is observed (B). Crick's angles and coiled coil radius calculation were made with Twister program (Strelkov and Burkhard, 2002).

Supplementary Fig. 2

Hydrophobic Cluster Analysis. Hydrophobic Cluster Analysis (Gaboriaud, 1987) Plot for HR1 and TM regions (A) and HR2, TM and cytoplasmic tail (B).