Peptidoglycan Metabolite Photoaffinity Reporters Reveal Direct Binding to Intracellular Pattern Recognition Receptors and Arf GTPases

The
peptidoglycan fragments γ-d-glutamyl-meso-diaminopimelic acid (iE-DAP) and muramyl-dipeptide
(MDP) are microbial-specific metabolites that activate intracellular
pattern recognition receptors and stimulate immune signaling pathways.
While extensive structure–activity studies have demonstrated
that these bacterial cell wall metabolites trigger NOD1- and NOD2-dependent
signaling, their direct binding to these innate immune receptors or
other proteins in mammalian cells has not been established. To characterize
these fundamental microbial metabolite-host interactions, we synthesized
a series of peptidoglycan metabolite photoaffinity reporters and evaluated
their cross-linking to NOD1 and NOD2 in mammalian cells. We show that
active iE-DAP and MDP photoaffinity reporters selectively cross-linked
NOD1 and NOD2, respectively, and not their inactive mutants. We also
discovered MDP reporter cross-linking to Arf GTPases, which interacted
most prominently with GTP-bound Arf6 and coimmunoprecipitated with
NOD2 upon MDP stimulation. Notably, MDP binding to NOD2 and Arf6 was
abrogated with loss-of-function NOD2 mutants associated with Crohn’s
disease. Our studies demonstrate peptidoglycan metabolite photoaffinity
reporters can capture their cognate immune receptors in cells and
reveal unpredicted ligand-induced interactions with other cellular
cofactors. These photoaffinity reporters should afford useful tools
to discover and characterize other peptidoglycan metabolite-interacting
proteins.