Secondary structure, dynamics, and architecture of the p7 membrane protein from hepatitis C virus by NMR spectroscopy

Abstract

P7 is a small membrane protein that is essential for the infectivity of hepatitis C virus. Solution-state NMR experiments on p7 in DHPC micelles, including hydrogen/deuterium exchange, paramagnetic relaxation enhancement and bicelle 'q-titration,' demonstrate that the protein has a range of dynamic properties and distinct structural segments. These data along with residual dipolar couplings yield a secondary structure model of p7. We were able to confirm previous proposals that the protein has two transmembrane segments with a short interhelical loop containing the two basic residues K33 and R35. The 63-amino acid protein has a remarkably complex structure made up of seven identifiable sections, four of which are helical segments with different tilt angles and dynamics. A solid-state NMR two-dimensional separated local field spectrum of p7 aligned in phospholipid bilayers provided the tilt angles of two of these segments. A preliminary structural model of p7 derived from these NMR data is presented.

Fig. 1

Two-dimensional ¹H-¹⁵N HSQC solution-state NMR spectrum of uniformly ¹⁵N-labeled HCV p7 in DHPC micelles at pH 4.0 and 50 °C. The spectrum is fully assigned with each of the backbone amides labeled.

Fig. 2

Assigned strip plots from the three-dimensional HNCA spectrum of p7. The connectivities of neighboring amino acids are shown, demonstrating how the protein resonances were assigned.

Fig. 3

¹H-¹⁵N HSQC spectra of uniformly ¹⁵N labeled HCV p7. (A) p7 in micelles in H₂O. (B) p7 in micelles in D₂O. (C) p7 in micelles with Mn²⁺-EDTA. (D) p7 in isotropic bicelles. The micelle samples contained 125 mM DHPC. For deuterium exchange experiments the sample was prepared in 75% D₂O. Mn²⁺-EDTA at a concentration of 10 mM was added to the sample for PRE experiments. The isotropic bicelle sample contained DMPC and DHPC (q = 0.5).

Fig. 4

Secondary structure plots of p7 in DHPC micelles. (A) ¹H-¹⁵N Residual Dipolar Couplings are plotted with the dipolar waves superimposed. (B) Cα chemical shift index values, (C) ¹H-¹⁵N Heteronuclear NOEs. (D) ¹H-¹⁵N HSQC intensities. (E) Relative intensity plots for a sample containing 75% D₂O (F) PRE data from a sample containing 10 mM Mn²⁺-EDTA (G) Intensities from an isotropic bicelle sample with q = 0.5 (G). A schematic representation of the seven structural elements of p7 is at the top of the Figure.

Fig. 5

Two-dimensional solid-state SAMMY spectrum of uniform ¹⁵N-labeled p7 in magnetically-aligned 14-O-PC/6-O-PC bilayers (q = 3.2, w/v = 28%). The spectrum was obtained at 42°C at 700 MHz. Simulated PISA wheels of tilt angles 10° and 25° are superimposed on the spectrum.

Fig. 6

Schematic structural representation of p7 in a phospholipid bilayer. Each of the seven structural segments is shown, with the four helical segments labeled A through D. The dynamic helices, A and C, are differentiated by their diagonal line pattern. The loop containing the two basic residues is shown facing the ER lumen while the N- and C-termini are shown exposed to the cytosol. The helical segments labeled B and D are shown tilted at 25 and 10°, respectively.