In the crystal structure 7YTX, the antagonist 3 displays a hydrogen bond with the backbone of residue Gly351, while simultaneously forming an ionic interaction with the side chain of residue Glu427. The antagonist is sterically hindered from exiting through the external path by the residues Lys350, Gly351 and Val520*, while the residues Glu427 and Tyr567* sterically hinder it from exiting the binding site through the internal path (Figure ). In some simulations, motion of the side chain of residue Tyr567* allowed the antagonist to exit the binding site through the internal pathway (Figure ). For cationic antagonists, unbinding via the internal path is facilitated by the motion of Tyr567*, explaining the preference of cationic antagonists for this path. When TLR8 is bound to neutral antagonists, Tyr567* forms a hydrogen bond with Glu427, which hinders the motion of Tyr567* and, therefore, makes ligand unbinding via the internal path more difficult (see below). This hydrogen bond is not formed in the structures of cationic antagonists due to the formation of an ionic interaction of Glu427 with the antagonists.