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J. Biol. Chem., Vol. 276, Issue 21, 18122-18129, May 25, 2001

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Structural Properties of Carnation Mottle Virus p7 Movement Protein and Its RNA-binding Domain^*

Marçal Vilar^§, Vicent Esteve, Vicente Pallás^¶, Jose F. Marcos, and Enrique Pérez-Payá^**

From the  Departament de Bioquímica i Biologia Molecular, Universitat de València, E-46100 Burjassot, València, Spain, the ^¶ Instituto de Biología Molecular y Celular de Plantas UPV-Consejo Superior de Investigaciones Científicas, Universidad Politécnica de València, Avda. de los Naranjos s/n, E-46022 València, Spain, and the  Departamento de Ciencia de los Alimentos, Instituto de Agroquímica y Tecnología de Alimentos, Consejo Superior de Investigaciones Científicas, Apartado de Correos 73, Burjassot, E-46100 València, Spain

Plant viral movement proteins (MPs) participate actively in the intra- and intercellular movement of RNA plant viruses to such an extent that MP dysfunction impairs viral infection. However, the molecular mechanism(s) of their interaction with cognate nucleic acids are not well understood, partly due to the lack of structural information. In this work, a protein dissection approach was used to gain information on the structural and RNA-binding properties of this class of proteins, as exemplified by the 61-amino acid residue p7 MP from carnation mottle virus (CarMV). Circular dichroism spectroscopy showed that CarMV p7 is an / RNA-binding soluble protein. Using synthetic peptides derived from the p7 sequence, we have identified three distinct putative domains within the protein. EMSA showed that the central region, from residue 17 to 35 (represented by peptide p7_17-35), is responsible for the RNA binding properties of CarMV p7. This binding peptide populates a nascent -helix in water solution that is further stabilized in the presence of either secondary structure inducers, such as trifluoroethanol and monomeric SDS, or RNA (which also changes its conformation upon binding to the peptide). Thus, the RNA recognition appears to occur via an "adaptive binding" mechanism. Interestingly, the amino acid sequence and structural properties of the RNA-binding domain of p7 seem to be conserved among carmoviruses and some other RNA-binding proteins and peptides. The low conserved N terminus of p7 (peptide p7_1-16) is unstructured in solution. In contrast, the highly conserved C terminus motif (peptide p7_40-61) adopts a -sheet conformation in aqueous solution. Alanine scanning mutagenesis of the RNA-binding motif showed how selected positive charged amino acids are more relevant than others in the RNA binding process and how hydrophobic amino acid side chains would participate in the stabilization of the protein-RNA complex.

The nucleotide sequence(s) reported in this paper has been submitted to the GenBank^TM/EMBL Data Bank with accession number(s) AJ304989.

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