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 pr otein of 15 kDa (p15) that is essential for the virus life cycle. It w as 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 recombina nt p15 was expressed in Escherichia coli and purified. Circular dichro ism spectroscopy showed that the peptide and the full-length protein c an assume an iv-helical conformation Analytical centrifugation allowed to determine that p15 assembles as a rod-shaped tetramer. Oxidative c ross-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 Lys(22) into Asp destabilized the tetramer and put forward the presence of a salt b ridge between Lys(22) and Asp(24) in a model building of the stalk. Th ese results suggest a model in which the stalk segment of p15 is locat ed at its N terminus, followed by a hinge that provides the space for presenting the C terminus for interactions with nucleic acids and/or p roteins.