Results of MD simulations of free diffusion of AX. (A) Relative frequencies of ligand pose (see color scale) as a function of the relative distance between the center of mass of AX and helix H4 (ΔD) and computed effective energies of binding (ΔGeffective). (B) Locations of the center of mass of AX (spheres) after 60 MD simulations of 400 nanosecond length each, with each simulation result colored differently. The black dashed line highlights all conformations that are bound to dimerization interface 1 with ΔDmin ≤ 0 Å, and the green dashed line highlights those with ΔDmin < 4 Å. The protein structure is shown as surface representation with the middle domain (not present during MD simulations) in orange and the CTD in white. In the panel, the structure is rotated by 180° around the y-axis. (C) Frequency of occupation of binding sites 1 (yellow), close to 1 (green; see definition in the main text), 2 (red), or 3 (blue) by AX across 60 MD simulations. (D) Binding mode model of AX. A representative conformation of AX bound to the CTD, extracted from the MD trajectory. Residues I688, I692, and M691 (gray spheres) bind to the side chain that distinguishes AX from 2. (E) An overlay of AX onto helix H5′ (Figure 1B-C) extracted from the crystal structure (PDB accession number 3Q6M57 ). In panels D and E, AX is depicted as blue sticks; hot spot amino acids I688, Y689, I692, and L69634 as gray sticks with Cβ atoms as magenta spheres; helix H5′ as a white cartoon with black backbone atoms; and the CTD in the left panel as surface representation, with all residues within 3 Å of AX colored in red. In panels A-C, 1, 2, and 3 denote the binding sites of AX, where 3 represents all binding sites besides 1 and 2.
