pH-sensitive residues in the p19 RNA silencing suppressor protein from carnation Italian ringspot virus affect siRNA binding stability

Abstract

Tombusviruses, such as Carnation Italian ringspot virus (CIRV), encode a protein homodimer called p19 that is capable of suppressing RNA silencing in their infected hosts by binding to and sequestering short-interfering RNA (siRNA) away from the RNA silencing pathway. P19 binding stability has been shown to be sensitive to changes in pH but the specific amino acid residues involved have remained unclear. Using constant pH molecular dynamics simulations, we have identified key pH-dependent residues that affect CIRV p19-siRNA binding stability at various pH ranges based on calculated changes in the free energy contribution from each titratable residue. At high pH, the deprotonation of Lys60, Lys67, Lys71, and Cys134 has the largest effect on the binding stability. Similarly, deprotonation of several acidic residues (Asp9, Glu12, Asp20, Glu35, and/or Glu41) at low pH results in a decrease in binding stability. At neutral pH, residues Glu17 and His132 provide a small increase in the binding stability and we find that the optimal pH range for siRNA binding is between 7.0 and 10.0. Overall, our findings further inform recent experiments and are in excellent agreement with data on the pH-dependent binding profile.

Figure 1

CIRV p19–siRNA complex. The complex has been rotated by 180° (PDBID: 1RPU).

Figure 2

Effects of pH on p19–siRNA binding. (A) Net charge of the holo and apo p19. (B) Total binding stability (white) and per residue contributions to binding stability (colors). Positive values increase binding stability (i.e., favors holo form) while negative values destabilizes binding (i.e., favors apo form). (C) pH-dependent dissociation constant profiles compared with experiment. CPHMD^shifted (red) is identical to CPHMD (yellow) except that it is shifted to the right by 1.5 pH units for comparison with experiment (black).

Figure 3

Individual contributions to binding stability at different pH ranges. Negative and positive contributions to binding stability will have and , respectively, for a given titratable residue. Only residues with are shown where is the Boltzmann constant and T is the temperature (298 K).

Figure 4

Conformational dynamics of key intraprotein and protein–RNA interactions. Lys–RNA and Glu–Arg distances correspond to salt bridge interactions. Trp–RNA distances and Cys SASA correspond to end-capping interactions and the solvent accessible surface area (SASA) for the cysteine sulfur atom, respectively. See Materials and Methods section for more detail.