Structural role of the conserved cysteines in the dimerization of the viral transmembrane oncoprotein E5.
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.
Windisch
Dirk
D
Karlsruhe Institute of Technology, DFG-Center for Functional Nanostructures, Institute of Organic Chemistry, Karlsruhe, Germany.
Hoffmann
Silke
S
Afonin
Sergii
S
Vollmer
Stefanie
S
Benamira
Soraya
S
Langer
Birgid
B
Bürck
Jochen
J
Muhle-Goll
Claudia
C
Ulrich
Anne S
AS
D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D052688:Bovine papillomavirus 1; D002462:Cell Membrane / Q000737:chemistry / Q000378:metabolism*; D002942:Circular Dichroism; D017124:Conserved Sequence; D003545:Cysteine; D004220:Disulfides / Q000737:chemistry; D006863:Hydrogen-Ion Concentration; D009682:Magnetic Resonance Spectroscopy; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D009856:Oncogene Proteins, Viral / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D055503:Protein Multimerization; D020836:Protein Structure, Quaternary; D013696:Temperature
D004220:Disulfides; D009856:Oncogene Proteins, Viral; C054304:oncogene protein E5, Bovine papillomavirus type 1; D003545:Cysteine