Figure 5.

Structure of SFTSV CBD and m⁷GTP binding. (A) The figure shows SFTSV CBD crystal structure in complex with an m⁷GTP. SFTSV CBD is presented as a ribbon diagram with the side chains of the two aromatic residues (F1703, Y1719) involved in stacking interaction with the m⁷GTP ligand shown as sticks. m⁷GTP is presented as lines and the surrounding electron density (2|Fo|-|Fc| omit map at 2.0σ) as gray mesh. (B) In a close-up of the m⁷GTP binding site, the protein is shown as ribbon diagram and side chains involved in m⁷GTP binding as well as the carbonyl oxygen of residue D1771 are presented as sticks. The m⁷GTP is shown as lines. The residues I1774, I1738, N1745, are involved in stabilizing the binding site cavity, D1771 (carbonyl oxygen), Q1707 and L1772 directly interact with m⁷GTP. A detailed list of interactions between the CBD and the m⁷GTP ligand is given in Supplementary Table S4 and a ligand plot in Supplementary Figure S13A. (C) Thermal stability of SFTSV CBD and influenza A virus PB2 CBD was tested in the presence and absence of different concentrations (2.5, 5.0, 7.5 and 10 mM) of m⁷GTP, m⁷GpppG, GTP and ATP. The melting temperatures (T_m) are presented as mean and standard deviations of three independent measurements (n = 3). (D) The affinity of SFTSV CBD for m⁷GTP and GTP was measured by isothermal titration calorimetry at 25°C. A representative titration curve is shown. Titrations were done three times with 150 μM SFTSV CBD in the cell and 5.0–6.5 mM m⁷GTP or GTP in the syringe. The upper panel shows the raw data, the lower panel the integrated data fitted to a single-site binding model with the stoichiometry fixed to 1. The dissociation constant K_D is given as a mean and standard deviation of three independent measurements for m⁷GTP.