HDX-MS Reveals A Loosened Gating Mechanism Of Q517H RIG-â…  Leads To Classic Singleton-Merten Syndrome

RIG-Ⅰ together with MDA5 and LGP2 belongs to the RLR family,and is an innate immune receptor recognizing the double-stranded RNA of virus during infections.The recognition of viral products by host’s immune receptors normally initiates a signal cascade to induce innate immune defenses and inflammations in the cell by pathogenassociated molecular patterns(PAMPs).RIG-Ⅰ is activated when binding to PAMP RNA,then interacts with the mitochondrial antiviral signaling protein(MAVS)to initiate a singal cascade,which ultimately leads to interferon and cell factor generation.RIG-Ⅰ signal transduction is essential to control the infection of many RNA viruses.However,recent studies have pointed out that some specific mutations in RIG-Ⅰ(including C268F/E373A/Q517H)are capable of leading to a rare immunogenetic disorder of Singleton-Merten syndrome(SMS).Variable phenotypes of SMS have been reported in the clinical diagnosis prominently characterized with psoriasis,acroosteolysis,and glaucoma,and with severe cases observed with dental dysplasia and tendon rupture.Atypical SMS RIG-Ⅰ mutants,such as C268 F and E373 A receptor mutations,are located in the ATPase cleft at the HEL1 RNA binding motifs I and II,forming a class of ATPase-deficient RIG-Ⅰ variants,allowing misrecognition of self-RNA.Recently,the new genotype Q517 Hof the gain-of-function variant of RIG-Ⅰ has been extended to another categoryunrelated to the ATP binding and hydrolysis functions of RIG-Ⅰ.Furthermore,the Q517 H variant renders a more severe phenotype of SMS.And at present,the molecular mechanism of Q517 H RIG-Ⅰ mediated autoimmune disorder remains unclear.Our study utilized hydrogen deuterium exchange mass spectrometry(HDX-MS),magnetic tweezer(MT)and biological experiments to analyze the molecular behaviors of Q517 H RIG-Ⅰ,and explain how it possesses constitutively active signaling that promotes aberrant interferon production.Our study showed that the activation of RIG-Ⅰ occurs in a carefully arranged order,and this process was visualized with the assistance of HDX-MS and magnetic tweezers.Under normal physiological conditions,RIG-Ⅰ is in an auto-inhibited state,and the CARDs(caspase activation recruitment domain)domains are inhibited through the CARD2-HEL2 i latch gate interface.After binding to PAMP RNA,the helicase domain of RIG-Ⅰ induces conformational changes,thereby releasing CARDs.Unlike PAMP RNA(3p10l),Cap2-10 l,which is a representative of self-RNA,weakens its binding affinity to WT RIG-Ⅰ due to the m7 G cap and N1,N2-2’-O-methylation,and then reducing CARDs release,and then may further resulting inthereduction and delay of the downstream signal of the receptor.However,gain-of-function mutation Q517 H allows the protein to evade the RNA surveillence checkpoint,thereby restoring the release of CARDs.For Q517 H RIG-Ⅰ,due to a lossened CARD2-HEL2 i latch gate in the apo state,CARDs is partially opened in the resting state.Q517 H RIG-Ⅰ can restore the binding to Cap2-10 l RNA similar as that of 3p10 l,thereby releasing the CARDs to an extent comparable with PAMP RNA,thereby activating downstream signaling pathways and stimulating unnecessary immune responses.In summary,our study proved that due to the mutation Q517 H,the RIG-Ⅰ gating mechanism CARD2-HEL2 i has been partially loosened,and the CARDs of RIG-Ⅰ are partially released from the self-inhibited state,leading to the errorenous recognition of Cap2 RNA,therefore leading to the abnormal expression of interferon and cause SMS.