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.肽聚糖片段γ-D-谷氨酰-内消旋二氨基庚二酸（γ-d-glutamyl-meso-diaminopimelic acid，iE-DAP）与胞壁酰二肽（muramyl-dipeptide，MDP）是一类微生物特异性代谢物，可激活细胞内模式识别受体并刺激免疫信号通路。尽管大量构效关系研究已证实，这些细菌细胞壁代谢物可触发NOD1与NOD2依赖的信号通路，但它们与这些固有免疫受体或哺乳动物细胞内其他蛋白质的直接结合尚未得到证实。为表征这些关键的微生物代谢物-宿主相互作用，我们合成了一系列肽聚糖代谢物光亲和探针（photoaffinity reporters），并评估了它们在哺乳动物细胞中与NOD1、NOD2的交联反应。研究显示，活性iE-DAP与MDP光亲和探针分别可选择性交联NOD1与NOD2，而非其失活突变体。我们还发现，MDP探针可与ADP核糖基化因子GTP酶（Arf GTPases）交联，这类GTP酶主要与GTP结合态的Arf6相互作用，并在MDP刺激后与NOD2发生共免疫沉淀。值得注意的是，与克罗恩病（Crohn’s disease）相关的功能丧失型NOD2突变体可消除MDP与NOD2及Arf6的结合。本研究证实，肽聚糖代谢物光亲和探针可在细胞中捕获其同源免疫受体，并揭示此前未被发现的配体诱导的与其他细胞辅因子的相互作用。这类光亲和探针有望成为发现并表征其他肽聚糖代谢物互作蛋白的实用工具。