Structural properties of carnation mottle virus p7 movement protein and its RNA-binding domain

Plant viral movement proteins (MPs) participate actively in the intra- and intercellular movement of RNA plant viruses to such an extent that MP dysfu nction impairs viral infection. However, the molecular mechanism(s) of thei r interaction with cognate nucleic acids are not well understood, partly du e to the lack of structural information. In this work, a protein dissection approach was used to gain information on the structural and RNA-binding pr operties of this class of proteins, as exemplified by the 61-amino acid res idue p7 MP from carnation mottle virus (CarMV). Circular dichroism spectros copy showed that CarMV p7 is an alpha/beta RNA-binding soluble protein. Usi ng synthetic peptides derived from the p7 sequence, we have identified thre e distinct putative domains within the protein. EMSA showed that the centra l region, from residue 17 to 35 (represented by peptide p(17-35)), is respo nsible for the RNA binding properties of CarMV p7, This binding peptide pop ulates a nascent alpha -helix in water solution that is further stabilized in the presence of either secondary structure inducers, such as trifluoroet hanol and monomeric SDS, or RNA (which also changes its conformation upon b inding to the peptide). Thus, the RNA recognition appears to occur via an " adaptive binding" mechanism. Interestingly, the amino acid sequence and str uctural properties of the RNA-binding domain of p7 seem to be conserved amo ng carmoviruses and some other RNA-binding proteins and peptides, The low c onserved N terminus of p7 (peptide p7(1-16)) is unstructured in solution. I n contrast, the highly conserved C terminus motif (peptide p7(40-61)) adopt s a beta -sheet conformation in aqueous solution. Alanine scanning mutagene sis of the RNA-binding motif showed how selected positive charged amino aci ds are more relevant than others in the RNA binding process and how hydroph obic amino acid side chains would participate in the stabilization of the p rotein-RNA complex.