Volume 25, Issue 1, Pages 53-65 (January 2017) Mechanism of Structural Tuning of the Hepatitis C Virus Human Cellular Receptor CD81 Large Extracellular Loop  Eva S. Cunha, Pedro Sfriso, Adriana L. Rojas, Pietro Roversi, Adam Hospital, Modesto Orozco, Nicola G.A. Abrescia  Structure  Volume 25, Issue 1, Pages 53-65 (January 2017) DOI: 10.1016/j.str.2016.11.003 Copyright © 2016 Elsevier Ltd Terms and Conditions

Structure 2017 25, 53-65DOI: (10.1016/j.str.2016.11.003) Copyright © 2016 Elsevier Ltd Terms and Conditions

Figure 1 Human CD81 Molecule (A) Overall view of the dimeric arrangement of hCD81LEL molecules in the current high-resolution P21 crystal and present as a common dimeric motif (oval symbol, 2-fold symmetry axis) across all six crystal forms (Table 1); one molecule is shown as a green cartoon and the other as dark gold cylinders schematically showing the five-helix bundle topology (from STRIDE secondary structure assignment: A = 116–136/116–136; B = 141–154/142–154; C = 166–172/163–172; D = 181–186/179–184; E = 190–199/190–199). (B) Chimerical montage of the dark gold hCD81LEL crystal structure in (A) and the predicted full tetraspanin CD81 model in white (PDB: 2AVZ; Seigneuret, 2006); represented as a cartoon with black arrows marking the direction of the transmembrane helices from the N terminus (N, blue) to the C terminus (C, red), and with black circles marking the SEL and helices C and D responsible for HCV binding, respectively; the schematic membrane separating the intra- and extracellular regions is shown in semi-transparent pale cream. Structure 2017 25, 53-65DOI: (10.1016/j.str.2016.11.003) Copyright © 2016 Elsevier Ltd Terms and Conditions

Figure 2 Conformational Variability of the hCD81LEL Head Subdomain (A) Graphic representation of the secondary structure assignment of the 15 X-ray structures: red helices, α helix; blue helices, 310 helix; yellow bar, turn or coil with the primary sequence from residues 159 to 190 and their correspondence to helices C and D at the top. The cardinal numbers identifying the molecules within each crystal form and the corresponding space groups are shown on the right and left sides of the secondary structure assignment graph, respectively. The colored circles nearby the molecule numbers associate with the conformation adopted by the CD81LEL head subdomain: red (closed), gold (intermediate), and green (open). The inset on the right shows in wall-eye stereoview the dynamism of helices C and D in the head subdomain as a superposition of the 15 hCD81LEL molecules (cartoon tube); the CD81LEL with the head subdomain in the closed conformation is colored in shades of red (from pink to red), in the intermediate conformation in shades of gold, and in the open conformation in shades of green. See also Movie S1. (B) Cylinder representation of hCD81LEL class representatives based on the radius of gyration (Rg) and the inter-helical angle (θ) between helices C and D, and color coded as in (A) with red, gold, and green representing the closed (mol-2), intermediate (mol-12), and open (mol-13) conformations, respectively. In the open conformer, the black circle marks the head-subdomain module rotation relative to the closed one; the two observed conformations of the C157-C175 disulfide bond are shown in stick representation (yellow/green) encircled by an oval dotted line. See also Figure S1. (C) View of the C157-C175 disulfide bridge in the CD81LEL molecules at 1.3 Å resolution. Top, wall-eye stereoview of the 2Fo – Fc electron density (white mesh, contoured at 1.0 σ) corresponding to the conformations I and II of C157-C175 in the open head subdomain (mol-13); bottom, as above but in the intermediate head subdomain (mol-12) only C175 shows side-chain mobility leading to disulfide bond conformation II. (D) Location of the four ionizable residues (in stick representation and color coded according to atom) with the two most shifted pKa values in hCD81LEL being hydrogen bond interacting residues H151 (−2.1 pH unit) and Y127 (3.9 pH unit) (labeled and at the core of the molecule), followed by the exposed residues D138 and E188 (−1.7 and 1.3 pH units, respectively); the yellow spheres identify C157 and C175 forming the S-S bridge. Inset, stereoview of the corresponding region. Structure 2017 25, 53-65DOI: (10.1016/j.str.2016.11.003) Copyright © 2016 Elsevier Ltd Terms and Conditions

Figure 3 Analysis of Molecular Contacts Difference in accessible surface area (Δ-ASA) by residue comprising the head subdomain for each molecule and for a given crystal form (from top-to-bottom in the same order as in Figure 2A). Values within each panel correspond to the total Δ-ASA. Red dots, green dots, and gold dots identify molecules in closed, open, and intermediate conformation, respectively. The red outline on the green dot of mol-13 indicates that helices C and D are almost parallel (θ = 134°). Structure 2017 25, 53-65DOI: (10.1016/j.str.2016.11.003) Copyright © 2016 Elsevier Ltd Terms and Conditions

Figure 4 Isopotential Surface and Hydrophobicity (A) Electrostatic isopotential surfaces of the three major head-subdomain conformations (as Figure 2B) contoured at levels of −5 kT/e (red) and 5 kT/e (blue) calculated at pH 7.4 (top) and at pH 4.0 (bottom). (B) Surface representation of the hydrophobicity distribution across the three head-subdomain conformations; the hydrophobic scale is shown in red with higher values in dark red. Structure 2017 25, 53-65DOI: (10.1016/j.str.2016.11.003) Copyright © 2016 Elsevier Ltd Terms and Conditions

Figure 5 Molecular Dynamics of hCD81LEL (A) Starting from an open conformation and a closed conformation (molecules 13 and 2), we followed the root-mean-square-fluctuation (RMSF) evolution of the head subdomain (light and dark blue) and the stalk subdomain (light and dark grey). The head subdomain shows most of the conformational variability. (B) Fluctuations of residues obtained after averaging 20 μs of MD trajectories. The moving elements of CD81LEL domain, color coded in yellow, pink, and red are mapped onto the structure. (C) Bi-dimensional distribution over the inter-helical angle (θ) and radius of gyration (Rg) space of the 2 × 105 structures generated during MD simulations. Most visited conformers, regions labeled with 1, 2, 3, and 4 on the map and representative structures on the right, reproduce the solved structures; the color scale on the far left shows the probability of a sampled structure belonging to a given point in the conformational space. Histograms (in red) on the top and right axes, representing the distribution of structural conformers, show that the closed conformations are overall favored. (D) Influence of the pH on the CD81LEL conformers. Distributions of structures observed at pH 4.0 (red) and pH 7.4 (grey) using the Rg criteria for structural classification showing that the increasingly open conformations (Rg ≥ 8 Å) are more favored at acidic pH. PDF is the probability density function. (E) Distribution of the S-S distance (between C157 and C175) derived from simulations at pH 7.4 (grey) and 4.0 (red), where the disulfide bridge was removed, starting from open (mol-14), intermediate (mol-12), and closed (mol-2) conformations (all together). The grey curve shows a narrower distribution of S-S distances supporting a more stable configuration at pH 7.4. See also Figure S2 and Movie S2. Structure 2017 25, 53-65DOI: (10.1016/j.str.2016.11.003) Copyright © 2016 Elsevier Ltd Terms and Conditions

Figure 6 Putative CD81 Mechanism of Action and HCV Cell-Entry Model (A) Cartoon of the FL-CD81 structural modularity with the two hinge movements between head/stalk and stalk/transmembrane regions with the head subdomain displaying structural variability. GLY, glycine. (B) Schematic model of HCV CD81-mediated cell entry. Top, HCV shown as a light-blue circle binding at the extracellular milieu to the closed head subdomain conformation of CD81 shown as red and light-red (multiple bindings could also occur); the complex then migrates to the tight junction where CD81 interacts with claudin-1 (not shown), preempting the uptake of the virus. Inset, the location of environmental-sensing residues at the core of the CD81LEL structure; shown as linked by the black arrow is the scenario in which, after internalization through the endocytic pathway, the different endosomal conditions (low pH and redox environment) would favor the head subdomain to rearrange into the open conformation enabling the HCV E1E2:CD81 complex to progress into the fusogenic state. The oligomeric state of FL-CD81 has been depicted as a dimer; the cell and the cell-membrane are shown in dark cream and pale cream, respectively (virus, receptor, and cell are not to scale). Structure 2017 25, 53-65DOI: (10.1016/j.str.2016.11.003) Copyright © 2016 Elsevier Ltd Terms and Conditions