Peptidoglycan Metabolite Photoaffinity Reporters Reveal Direct Binding to Intracellular Pattern Recognition Receptors and Arf GTPases.
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ABSTRACT: The peptidoglycan fragments ?-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.
SUBMITTER: Wang YC
PROVIDER: S-EPMC6943928 | biostudies-literature | 2019 Mar
REPOSITORIES: biostudies-literature
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