The open reading frame III product of cauliflower mosaic virus forms a tetramer through a N-terminal coiled-coil.
The open reading frame III product of cauliflower mosaic virus is a protein of 15 kDa (p15) that is essential for the virus life cycle. It was shown that the 34 N-terminal amino acids are sufficient to support protein-protein interaction with the full-length p15 in the yeast two-hybrid system. A corresponding peptide was synthesized and a recombinant p15 was expressed in Escherichia coli and purified. Circular dichroism spectroscopy showed that the peptide and the full-length protein can assume an alpha-helical conformation. Analytical centrifugation allowed to determine that p15 assembles as a rod-shaped tetramer. Oxidative cross-linking of N-terminal cysteines of the peptide generated specific covalent oligomers, indicating that the N terminus of p15 is a coiled-coil that assembles as a parallel tetramer. Mutation of Lys22 into Asp destabilized the tetramer and put forward the presence of a salt bridge between Lys22 and Asp24 in a model building of the stalk. These results suggest a model in which the stalk segment of p15 is located at its N terminus, followed by a hinge that provides the space for presenting the C terminus for interactions with nucleic acids and/or proteins.
Leclerc
D
D
Friedrich Miescher Institut, P. O. Box 2543, CH-4002 Basel, Switzerland.
Burri
L
L
Kajava
A V
AV
Mougeot
J L
JL
Hess
D
D
Lustig
A
A
Kleemann
G
G
Hohn
T
T
D000595:Amino Acid Sequence; D001483:Base Sequence; D001704:Biopolymers; D017796:Caulimovirus / Q000737:chemistry*; D002850:Chromatography, Gel; D017931:DNA Primers; D008969:Molecular Sequence Data; D016296:Mutagenesis; D016366:Open Reading Frames; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D055672:Static Electricity; D014764:Viral Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
D001704:Biopolymers; D017931:DNA Primers; D011994:Recombinant Proteins; D014764:Viral Proteins; C115486:p15 protein, Cauliflower mosaic virus