Small molecule inhibitors reveal an indispensable scaffolding role of RIPK 2 in NOD 2 signaling

Abstract

RIPK2 mediates inflammatory signaling by the bacteria‐sensing receptors NOD1 and NOD2. Kinase inhibitors targeting RIPK2 are a proposed strategy to ameliorate NOD‐mediated pathologies. Here, we reveal that RIPK2 kinase activity is dispensable for NOD2 inflammatory signaling and show that RIPK2 inhibitors function instead by antagonizing XIAP‐binding and XIAP‐mediated ubiquitination of RIPK2. We map the XIAP binding site on RIPK2 to the loop between β2 and β3 of the N‐lobe of the kinase, which is in close proximity to the ATP‐binding pocket. Through characterization of a new series of ATP pocket‐binding RIPK2 inhibitors, we identify the molecular features that determine their inhibition of both the RIPK2‐XIAP interaction, and of cellular and in vivo NOD2 signaling. Our study exemplifies how targeting of the ATP‐binding pocket in RIPK2 can be exploited to interfere with the RIPK2‐XIAP interaction for modulation of NOD signaling.

A regulatory interface on RIPK2 is required for XIAP binding and NOD signaling activity

“…Alternatively, XIAP could bind somewhere else, and mutation of K209 and I212 might lead to conformational changes that have an impact on the structure or orientation of the interaction interface. A region in the N-lobe of RIPK2, in particular residues R36 and R41, was identified as a critical interaction region for the BIR2 of XIAP [20]. We used the reported R41L mutation as a control for our assay and confirmed the impaired binding of XIAP BIR2 to this mutant.…”

“…Wild-type RIPK2 was strongly enriched by XIAP-BIR2 pulldown, and I212A was even more abundant, correlating with the increased stimulus dependent ubiquitination of this mutant and increased cytokine secretion compared to wild-type RIPK2. Binding of K209R and I212D to the BIR2 of XIAP was, however, drastically reduced and comparable to R41L, a RIPK2 mutant previously shown to have impaired binding to XIAP [20]. This binding site for XIAP, involving R41 and R36, is in the N-lobe of the RIPK2 kinase domain and therefore quite separate (approximately 40 Å) from the C-lobe pocket, suggesting that XIAP may bind multiple sites on RIPK2.…”

“…The kinase activity of RIPK2 was initially reported to be required for signal transduction and for critical autophosphorylation of RIPK2 on S176 in the activation loop of the kinase domain [17] and Y474 in its CARD [18]. However, recent studies suggest that RIPK2 kinase activity is dispensable for NF-κB activation and cytokine production [19,20]. Furthermore, it has been established that NOD signaling relies on ubiquitination of RIPK2 [21].…”

“…RIPK2 has been established as a potential drug target, particularly in inflammatory bowel disease, and RIPK2-targeting kinase inhibitors have been developed [24,26,28]. Recent studies showed that the inhibition of NOD signaling is not directly due to the inhibition of the kinase function of RIPK2, but rather by the disruption of the RIPK2-XIAP interaction [19,20,26]. This has led to the hypothesis that protein-protein interaction inhibitors could be used to treat NOD-driven diseases and highlights the need for a detailed understanding of posttranslational modifications on RIPK2.…”

Evidence of a noncoding transcript of theRIPK2gene overexpressed in head and neck tumor

“…In fact, literature has already referred that a mild hypothermic condition appears to induce or to inhibit synthesis of specific proteins when compared with control cells, an effect that is cell line dependent (reviewed by [33]. It’s well known that both prokaryotic and eukaryotic organisms respond to cold stress reducing transcription, translation, and metabolic processes, except in the case of cold-shock proteins [34].…”