Abstract
Innate immune receptors NOD1 and NOD2 are activated by bacterial peptidoglycans leading to recruitment of adaptor kinase RIP2, which, upon phosphorylation and ubiquitination, becomes a scaffold for downstream effectors. The kinase domain (RIP2K) is a pharmaceutical target for inflammatory diseases caused by aberrant NOD2-RIP2 signalling. Although structures of active RIP2K in complex with inhibitors have been reported, the mechanism of RIP2K activation remains to be elucidated. Here we analyse RIP2K activation by combining crystal structures of the active and inactive states with mass spectrometric characterization of their phosphorylation profiles. The active state has Helix αC inwardly displaced and the phosphorylated Activation Segment (AS) disordered, whilst in the inactive state Helix αC is outwardly displaced and packed against the helical, non-phosphorylated AS. Biophysical measurements show that the active state is a stable dimer whilst the inactive kinase is in a monomer-dimer equilibrium, consistent with the observed structural differences at the dimer interface. We conclude that RIP2 kinase auto-phosphorylation is intimately coupled to dimerization, similar to the case of BRAF. Our results will help drug design efforts targeting RIP2 as a potential treatment for NOD2-RIP2 related inflammatory diseases.
Highlights
NOD1 and NOD2 (Nucleotide-binding oligomerization domain-containing proteins 1 and 2) are cytosolic receptors of the innate immune system, which respond to intracellular fragments of the bacterial peptidoglycans, D-glutamyl-meso-diaminopimelic acid and muramyl dipeptide (MPD), respectively [1,2,3,4,5]
In the present study we provide a comprehensive structural description of the kinase domain of Receptor Interacting protein kinase 2 (RIP2) in its active and inactive states
Together with mass spectrometry (MS), analytical ultra-centrifugation (AUC) data and mutagenesis experiments, our results provide an overview of the conformational changes necessary to activate RIP2K
Summary
NOD1 and NOD2 (Nucleotide-binding oligomerization domain-containing proteins 1 and 2) are cytosolic receptors of the innate immune system, which respond to intracellular fragments of the bacterial peptidoglycans, D-glutamyl-meso-diaminopimelic acid (iE-DAP) and muramyl dipeptide (MPD), respectively [1,2,3,4,5]. NOD receptors oligomerise and recruit an adaptor protein, Receptor Interacting protein kinase 2 (RIP2), through interactions of their respective CARD domains [6]. RIP2 contains an N-terminal dualspecificity ser/tyr kinase domain (RIP2K), which belongs to the TKL kinase family (Tyrosine kinase-like) [4, 7]. RIP2 undergoes auto-phosphorylation and K-63 linked ubiquitination at Lys209, becoming a scaffolding protein for downstream effectors.
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