The p7 protein of hepatitis C virus forms structurally plastic, minimalist ion channels

Abstract

Hepatitis C virus (HCV) p7 is a membrane-associated oligomeric protein harboring ion channel activity. It is essential for effective assembly and release of infectious HCV particles and an attractive target for antiviral intervention. Yet, the self-assembly and molecular mechanism of p7 ion channelling are currently only partially understood. Using molecular dynamics simulations (aggregate time 1.2 µs), we show that p7 can form stable oligomers of four to seven subunits, with a bias towards six or seven subunits, and suggest that p7 self-assembles in a sequential manner, with tetrameric and pentameric complexes forming as intermediate states leading to the final hexameric or heptameric assembly. We describe a model of a hexameric p7 complex, which forms a transiently-open channel capable of conducting ions in simulation. We investigate the ability of the hexameric model to flexibly rearrange to adapt to the local lipid environment, and demonstrate how this model can be reconciled with low-resolution electron microscopy data. In the light of these results, a view of p7 oligomerization is proposed, wherein hexameric and heptameric complexes may coexist, forming minimalist, yet robust functional ion channels. In the absence of a high-resolution p7 structure, the models presented in this paper can prove valuable as a substitute structure in future studies of p7 function, or in the search for p7-inhibiting drugs.

Figure 1. Oligomeric p7 models constructed from the NMR p7 monomer , using symmetry operations.

Box A: (a) Heptamer A, (b) Hexamer A; Box B: (a) Heptamer B, (b) Hexamer B, (c) pentamer, (d) tetramer; Box C: Hexamer C. The models in box A were constructed so that the monomers extend radially away from the center, similar to the model presented earlier . In contrast, the models in box B were built such that the monomers optimize the contacts between their mutual surfaces, consistent with the models described in two prior studies , . The model in box C was built to be similar to, but have the opposite handedness of Hexamer B.

Figure 2. Time-evolution of the structural parameters of the oligomers.

Distance root-mean square deviation (RMSD) with respect to the starting conformation of the p7 channel, computed over backbone atoms (A), and solvent-accessible surface area (SASA) per subunit (B).

Figure 3. Molecular assemblies and pore-radius profiles of p7 Hexamer B (A) and Heptamer B (B) pores in a POPC bilayer.

Left panels: side views of Hexamer B and Heptamer B (ribbon representation) with water molecules inside the central pore represented as van der Waals spheres (red and grey). The side chains of F25 (in green) and I32 (in yellow) are represented as sticks. The model POPC bilayer is described by means of a stick representation (light grey), with phosphorus and nitrogen atoms displayed as glossy orange and green van der Waals spheres, respectively. Right panels: radial profiles depicting the width of the pore along the longitudinal axis of the complex were calculated using the program Hole . The red line is positioned at 1.4 Å, the approximate radius of a water molecule. The green and yellow lines mark the approximate positions of F25 and I32, respectively. In the Hexamer B plot (A), the solid line represents the radial profile before the opening of the pore and highlights a constriction at the level of F25. The dotted line denotes the radial profile after the pore has opened (the protrusion near z = 25 Å is the result of association between the N-terminal tails of the six subunits in the solvent below the membrane, and does not represent a blockage of the pore).

Figure 4. Conformation of the p7 hexamer in short– and long–tail lipid environments.

(A) Hexamer B model (ribbon representation) fitted into the EM envelope of Luik et al . (B) Side views of the EM-fitted hexamer model in the DHPC and the POPC lipid bilayers (for details and color code, see legend of Figure 3). (C) Tilt angles of TM1 and TM2 helices calculated for all models (for color code of oligomers, see legend of Figure 2). Relaxation of the model placed in POPC towards an upright conformation akin to that of Hexamer B is recognized through lower values of the TM1 and TM2 tilt angles.