Structural role of the conserved cysteines in the dimerization of the viral transmembrane oncoprotein E5

Abstract

The E5 oncoprotein is the major transforming protein of bovine papillomavirus type 1. This 44-residue transmembrane protein can interact with the platelet-derived growth factor receptor β, leading to ligand-independent activation and cell transformation. For productive interaction, E5 needs to dimerize via a C-terminal pair of cysteines, though a recent study suggested that its truncated transmembrane segment can dimerize on its own. To analyze the structure of the full protein in a membrane environment and elucidate the role of the Cys-Ser-Cys motif, we produced recombinantly the wild-type protein and four cysteine mutants. Comparison by circular dichroism in detergent micelles and lipid vesicular dispersion and by NMR in trifluoroethanol demonstrates that the absence of one or both cysteines does not influence the highly α-helical secondary structure, nor does it impair the ability of E5 to dimerize, observations that are further supported by sodium dodecylsulfate polyacrylamide gel electrophoresis. We also observed assemblies of higher order. Oriented circular dichroism in lipid bilayers shows that E5 is aligned as a transmembrane helix with a slight tilt angle, and that this membrane alignment is also independent of any cysteines. We conclude that the Cys-containing motif represents a disordered region of the protein that serves as an extra covalent connection for stabilization.

Figure 1

Nomenclature and sequences of the native E5 (E5-CSC) and the four cysteine mutants used in our study. The gray box represents the putative transmembrane segment between Leu⁷ and Leu²⁹. At the N-terminus, there is an additional glycine due to hydroxylamine cleavage. The amino acid substitutions between positions 37 and 39 of the different E5-mutants are highlighted.

Figure 2

(A) Chromatographic analysis of E5-CSC and the cysteine-free mutant E5-ASA. In the HPLC trace, the wild-type protein eluted as two distinct fractions dominated by monomers (retention time of 12 min) or dimers (retention time of 18 min). The molecules in the dimer fraction could be converted into momomers by reduction of the disulfide bridges with TCEP. E5-ASA consisted mostly of monomers, as expected. (B and C) SDS-PAGE of the dimeric (B) and monomeric (C) fractions under nonreducing conditions. Lanes represent a molecular weight marker (a), E5-CSC (b), E5-ASC (c), E5-CSA (d), E5-ACA (e), and E5-ASA (f).

Figure 3

CD spectra of E5-CSC and mutants in detergent micelles and liposomes. (A) Aqueous solution of 10 mM LPPC micelles at 20°C, showing a predominantly α-helical conformation of the proteins. (Inset) Measured CD spectra normalized to the maximum ellipticity at 194 nm. (B) DMPC/LMPC vesicle suspensions (pH 3) with a protein/lipid ratio of 1:300 (mol/mol) measured above the lipid phase transition at 30°C.

Figure 4

CD spectra showing the pH and temperature dependence of E5-CSC, acquired in 10 mM DPC micellar solution. By heating to 90°C and cooling back to 20°C, the aggregated protein at pH 7 could be converted into almost the same regular helical structure as the acidified sample maintained at pH 3.

Figure 5

OCD spectra of E5-CSC and mutants in oriented lipid bilayers of DMPC/LMPC, acquired at 30°C and 97% relative humidity. (Inset) The spectra are normalized to the same intensity (225 nm) to illustrate the similarity in the lineshapes.

Figure 6

Superposition of the ¹H¹⁵N-HSQC spectra of wild-type E5-CSC (black) and mutants (red) E5-ASC (A), E5-CSA (B), E5-ACA (C), and E5-ASA (D) in 80% TFE.

Figure 7

(A) Proposed model of the membrane-inserted structure of E5. The gray box represents the putative transmembrane segment that forms the regular helix fraction (solid outline), which is flanked by distorted helical regions (dotted outline) that extend beyond the lipid bilayer. The cysteines are located in the adjacent unstructured stretch near the C-terminus. (B) Three-dimensional scheme of the E5 dimer in a lipid bilayer based on the model in A. The regular helix parts are shown as red and the distorted helical regions as pale red in the cylinders, and the unstructured parts are shown as black lines. The cysteines are arranged to allow parallel disulfide bridges between Cys³⁷-Cys³⁷ and Cys³⁹-Cys³⁹. The sketch was made using POV-Ray (Persistence of Vision Raytracer).