Structural basis of ligand interactions of the large extracellular domain of tetraspanin CD81

Abstract

Hepatitis C virus (HCV) causes chronic liver disease, cirrhosis, and primary liver cancer. Despite 130 million people being at risk worldwide, no vaccine exists, and effective therapy is limited by drug resistance, toxicity, and high costs. The tetraspanin CD81 is an essential entry-level receptor required for HCV infection of hepatocytes and represents a critical target for intervention. In this study, we report the first structural characterization of the large extracellular loop of CD81, expressed in mammalian cells and studied in physiological solutions. The HCV E2 glycoprotein recognizes CD81 through a dynamic loop on the helical bundle, which was shown by nuclear magnetic resonance (NMR) spectroscopy to adopt a conformation distinct from that seen in crystals. A novel membrane binding interface was revealed adjacent to the exposed HCV interaction site in the extracellular loop of CD81. The binding pockets for two proposed inhibitors of the CD81-HCV interaction, namely, benzyl salicylate and fexofenadine, were shown to overlap the HCV and membrane interaction sites. Although the dynamic loop region targeted by these compounds presents challenges for structure-based design, the NMR assignments enable realistic screening and validation of ligands. Together, these data provide an improved avenue for developing potent agents that specifically block CD81-HCV interaction and also pave a way for elucidating the recognition mechanisms of diverse tetraspanins.

Fig 1

Recombinant CD81-LEL is functionally and conformationally intact. (A) Neutralization assay of HCV infection with CD81-LEL domain proteins. HCVcc infections of Huh-7 cells were performed in the presence of the indicated concentrations of proteins. At 2 days postinfection, cells were lysed and processed as described in Materials and Methods. (B) Profiles for the area under the concentration-time curve (AUC) are shown for CD81-LEL (continuous line) and CD81-LEL^HEK (dotted line), indicating dimeric states based on the protein sedimentation coefficients of 2.2 and 2.1, respectively (corresponding to molecular masses of 22.3 and 22.6 kDa). (C) Far-UV CD spectra of CD81-LEL (continuous line) and CD81-LEL^HEK (dotted line) show characteristic α-helical protein profiles with double negative minima, at 208 and 222 nm.

Fig 2

¹H-¹⁵N-HSQC spectrum of 1 mM CD81-LEL. All assigned backbone amide resonances are labeled. The inset shows a magnified region from a highly overlapped area of the spectrum.

Fig 3

(A) Summary of secondary structure predictions for the CD81-LEL domain, obtained using CSI, TALOS+, and RCI. In the consensus CSI graph, −1 indicates an α-helical tendency and +1 represents a β-strand or loops. Backbone dihedral angles (phi and psi) were calculated using TALOS+. Phi (φ) and psi (ψ) angles are shown in black and red, respectively. The middle panel shows the S² values for the backbone amide groups predicted by the RCI approach. α-Helical secondary structures are represented with open cylinders (X-ray) and closed cylinders (NMR). The helix D not observed in solution is shown as a dotted open cylinder. Solvent-accessible residues based on backbone amide NOEs on water resonances from a 3D ¹⁵N-NOESY-HSQC spectrum are indicated by dark circles. (B) 2D strips from the 3D ¹⁵N-separated NOESY-HSQC spectra of CD81-LEL, showing NOE sequential connectivity (dotted lines) in the SNLFK motif residues at the indicated ¹⁵N slices (in parentheses).

Fig 4

Micelle binding interface of CD81-LEL. (A) Residues that showed significant (>85%) decreases in ¹H and ¹⁵N cross-peak intensities in HSQC spectra of CD81-LEL upon increasing the DPC concentration from 400 mM to 800 mM are labeled and shown in green. (B) Micelle binding interface residues were mapped onto the surface of a CD81-LEL dimer and colored green. The dimer symmetry axis that separates the monomers is indicated by a dotted line, with the second monomer shown with 50% transparency.

Fig 5

Chemical shift-based mapping of inhibitor binding to ¹⁵N-CD81-LEL. (A) Chemical shift perturbations (Δδ) of cross peaks from ¹H-¹⁵N-HSQC spectra of CD81-LEL in the presence of 1.6 mM benzyl salicylate. The cross peaks of residues undergoing significant perturbation (mean + 1 SD) are labeled and color coded according to Δδ values, as follows: red, >16 Hz; orange, 12 to 16 Hz; and yellow, 8 to 12 Hz. The chemical structure of benzyl salicylate is shown as an inset. Residues that interact with benzyl salicylate were mapped onto a monomeric ribbon structure (B) and a dimeric surface structure (C) of CD81-LEL. (D) Chemical shift perturbation of CD81-LEL residues in the presence of 1.6 mM fexofenadine. Cross peaks of residues undergoing significant perturbation (mean + 1 SD) are labeled and color coded according to Δδ values, as follows: red, >14 Hz; orange, 10 to 14 Hz; and yellow, 7 to 10 Hz. The chemical structure of fexofenadine is shown as an inset. Residues that interact with fexofenadine were mapped onto a monomeric ribbon structure (E) and a dimeric surface structure (F) of CD81-LEL. The dimer symmetry axis that separates monomers is indicated by a dotted line, with the second monomer shown in 50% transparency.

Fig 6

Model of CD81-LEL monomer indicating the ligand binding residues identified in this study, along with the previously reported surface. (Left) CD81 monomer obtained using the crystal structure of CD81-LEL (PDB accession no. 1IV5.pdb) with the SEL (orange β-strand) modeled from the full-length CD81 model (PDB accession no. 2AVZ.pdb), highlighting the E2 binding SNLFK motif (red), the DPC binding interface (green), and the previously reported Plasmodium sporozoite interacting VVDDD motif (pink) (54). (Right) Rotation of 120° around the vertical axis of the model on the left, highlighting the proximity of HCV E2 and membrane binding interfaces.