The cellular receptor to human rhinovirus 2 binds around the 5-fold axis and not in the canyon: a structural view

Abstract

Human rhinovirus serotype 2 (HRV2) belongs to the minor group of HRVs that bind to members of the LDL-receptor family including the very low density lipoprotein (VLDL)-receptor (VLDL-R). We have determined the structures of the complex between HRV2 and soluble fragments of the VLDL-R to 15 A resolution by cryo-electron microscopy. The receptor fragments, which include the first three ligand-binding repeats of the VLDL-R (V1-3), bind to the small star-shaped dome on the icosahedral 5-fold axis. This is in sharp contrast to the major group of HRVs where the receptor site for ICAM-1 is located at the base of a depression around each 5-fold axis. Homology models of the three domains of V1-3 were used to explore the virus-receptor interaction. The footprint of VLDL-R on the viral surface covers the BC- and HI-loops on VP1.

Fig. 1. Schematic diagram of VLDL-R. A recombinant soluble fragment protein was expressed encompassing repeats 1–3 fused via their N-terminus to maltose binding protein. Repeats 1–3 were cleaved from the fusion partner by factor Xa. The amino acid sequence of part of the fusion protein and of the individual repeats 1–3 are given, and the cleavage sites for factor Xa and for an unidentified bacterial protease (BP) are indicated. Acidic amino acids are shaded black and cysteines are shaded grey. The molecular weight of the entire fusion protein is 59 kDa and of V1–3, including the His₆ tag, is 17 kDa. Acidic amino acids involved in Ca²⁺ coordination are indicated (#).

Fig. 2. One asymmetric unit of the icosahedral capsid is depicted on the X-ray map of HRV2 limited to 15 Å resolution in (A). Electron micrographs of frozen hydrated native HRV2 (B), the HRV2–V1–3 complex (C) and the HRV2–MBP-V1–3 complex (D) are presented. Arrowheads indicate protuberances, which are probably MBP (D). Stereo views of the X-ray map of HRV2 (E) limited to 15 Å resolution with a Debye–Waller factor of 500 Å², and the reconstructed cryo-electron microscopy map of HRV2 (F) to 15 Å resolution viewed down the 2-fold axis, show the same features. Stereo views of the reconstructed HRV2–V1–3 (G) and HRV2–MBP-V1–3 (H) complexes, also viewed down a 2-fold axis. A ‘crown’ of receptor molecules is seen on each 5-fold axis.

Fig. 3. Difference maps showing V1–3 (A–C) and MBP-V1–3 (D–F), extracted from the maps of the complex with HRV2, are shown for three different thresholds. In both difference maps, a small protuberance is visible at low contour (arrows in C and F). (G) A view, down the 5-fold axis, of the native HRV2 reconstruction at the same scale and orientation for reference. (H) A central section of the HRV2–MBP-V1–3 density map. The weak density attributed to the MBP fusion protein is marked with a black arrow. The density attributed to the receptor is arrowed in white. The scale bars represent 5 nm.

Fig. 4. Stereo views of difference maps showing V1–3 (A) and MBP-V1–3 (B) viewed down a 2-fold axis. Only the front half of both difference maps are shown for a radius from 145 to 185 Å and the noise has not been removed. The contour level is similar to that in Figure 2. Note the ‘crown’ has extensions down the sides of the dome on the 5-fold axis. The scale bar represents 10 nm.

Fig. 5. Stereo views of the fit of HRV2 and V1 and V2 in the cryo-electron microscope map of the HRV2–V1–3 complex. The Cα backbones of HRV2 VP1 and the receptor domains V1 and V2 are coloured blue, white and yellow, respectively. The electron microscope map is depicted in green. The footprint of V1–3 on HRV2 is shown in (A). On two VP1 the footprint of V1–3 is coloured in red, and residues Lys81 and ThrGluLys 222–224 are represented in detail in yellow. The asterisk indicates the region of density that is not filled by the native HRV2 or by V1 or V2 in our model. Fits of the homology structures of V1 and V2 viewed down a 5-fold axis (B) and perpendicular to a 5-fold axis (C) are shown.

Fig. 6. ‘Road map’ showing the surface of HRV2 with different shades of grey representing the distance from the viral centre (bottom). Enlarged view of the receptor ‘foot print’ on HRV2 (top). The figure was created with the program ‘RoadMap’ (Chapman, 1993) using the HRV2 PDB co-ordinate file (1FPN). The 5-fold axis of symmetry is indicated by a pentagon. The whole triangle represents one icosahedral asymmetric unit projected on to a plane perpendicular to the 2-fold axis.

Fig. 7. Schematic diagram of the VLDL-R soluble fragment bound to HRV2. An arrow indicates the 5-fold axis. In this model only the first two ligand-binding domains interact with the capsid.