UC San Diego
The NMR Structure and Oligomerization State of the Membrane Associated p7 Protein from Hepatitis C Virus
- Author(s): Dawson, Lindsay A.
- et al.
The Hepatitis C viral (HCV) protein p7 has been of interest in the HCV community as a potential drug target for the treatment of hepatitis for over two decades. p7 is considered to be a viroporin, shown to oligomerize and induce channel activity, allowing channel-blocking compounds to target the porin as an effective antiviral treatment plan. High-resolution structural data is absolutely vital to a rational drug design. This includes determining not only preferred oligomerization state but also determining a biologically relevant, high-resolution structure of p7. The backbone structure of p7 was determined in detergent micelles using solution NMR and conveys condensed structure due to the limited breadth of the membrane environment in detergent micelles. Here, both the solution and the solid-state monomeric structure of p7 coupled with the elucidation of the oligomerization state are the focus of our studies. The solution structure was determined with the help of mutational studies to dampen dynamics of unresolved regions of the protein structure. A single point mutation of an absolutely conserved residue, W48A, is used to help refine the solution structure. Additionally, interaction studies are observed for the transmembrane region of p7 and NS2, a nonstructural p7- interactting HCV protein. Drug-binding studies are conducted to map specific residues involved in binding known channel-blocking compounds, amantadine, NN-DNJ and HMA. The solid-state backbone structure is determined for p7 reconstituted in proteoliposomes using rotationally aligned (RA) solid-state NMR. Various methods of chemical cross-linking are carried out to establish an optimal method for determining oligomerization state. p7 is cross- linked in proteoliposomes using an amine-reactive cross- linker and products were visualized using silver-stained SDS-PAGE. A modified native PAGE technique using perfluorooctanoic acid (PFO) is applied to study the oligomerization state in a mild detergent native-like state. Unmodified p7 is incorporated into nanodiscs and a light sensitive chemical cross-linker is used to characterize preferred oligomerization state. In addition to these studies, the expression and purification of a separate genotype, 2a, subtype JFH-1 p7 is optimized for a structural comparison to J4 subtype