The C-terminal domain of the 2b protein of Cucumber mosaic virus is stabilized by divalent metal ion coordination.

The main function of the 2b protein of Cucumber mosaic virus (CMV) is binding permanently the double stranded siRNA molecules in the suppression process of post-transcriptional gene silencing (PTGS). The crystal structure of the homologue Tomato aspermy virus (TAV) 2b protein is known, but without the C-terminal domain. The biologically active form is a tetramer: four 2b protein molecules and two siRNA duplexes. Regarding the complete 2b protein structure, we performed a molecular dynamics (MD) simulation of the whole siRNA-2b ribonucleoprotein complex. Unfortunately, the C-terminal domain is proved to be partially unstructured. Multiple sequence alignment showed a well conserved motif between residues 94 and 105. The negatively charged residues of the C-terminal domain are supposed to take part in coordination of a divalent metal ion and stabilize the three-dimensional structure of the C-terminal domain. MD simulations were performed on the detached C-terminal domains (aa 65-110). 0.15 M MgC₂, CaCl₂, FeCl₂ and ZnCl₂ salt concentrations were used in the screening simulations. Among the tested divalent metal ions Mg²⁺ proved to be very successful because Asp95, Asp96 and Asp98 forms a quasi-permanent Mg²⁺ binding site. However the control computations have resulted in any (at least) divalent metal ion remains in the binding site after replacement of the bound Mg²⁺ ion. A quadruple mutation (Rs2DDTD/95-98/AAAA) was introduced into the position of the putative divalent metal ion binding site to analyze the biological relevance of molecular modeling derived hypothesis. The plant inoculation experiments proved that the movement of the mutant virus is slower and the symptoms are milder comparing to the wild type virus. These results demonstrate that the quadruple mutation weakens the stability of the 2b protein tetramer-siRNA ribonucleoprotein complex.