Membrane targeting enhances muramyl dipeptide binding to NOD2 and Arf6–GTPase in mammalian cells

To further understand the mechanisms of muramyl dipeptide (MDP) sensing by NOD2, we evaluated key properties involved in the formation of the Arf6-MDP-NOD2 complex in mammalian cells. We found that the conserved Arf aromatic triad is crucial for binding to MDP-NOD2. Mutation of Arf6 N-myristoylation and NOD2 S-palmitoylation also abrogated the formation of the Arf6-MDP-NOD2 complex. Notably, lipid-modified MDP (L18-MDP) increased Arf6-NOD2 assembly. Our results indicate recruitment of Arf6 may explain enhanced activity of lipidated MDP analogues and membrane targeting may be important in developing next-generation NOD2 agonists.