Structural And Functional Studies Of CBM Complex In Antigen Receptor-induced NF-κB Pathway

The CBM complex is a central signalling component that has a critical role in lymphocyte activation and proliferation through the regulation of nuclear factor-κB (NF-kB) pathway following antigen receptor stimulation.The activation of transcription factors of NF-κB family play a key role in both innate and adaptive immunity by modulating the transcription of various genes that relate to immune response. CBM complex consists of CARMA1, BCL10and MALT1. As a scaffold protein, CARMA1recruits two signaling proteins BCL10and MALT1in response to stimulus. CARMA1and BCL10associate through the CARD/CARD interaction that is initiated by a stimulation-dependent conformational change in CARMA1, adapter molecule BCL10forms a complex with MALT1constitutively, it is demonstrated that CARMA1interacts with MALT1via a Coiled-Coil (CC) domain, which may contributes to stabilizing the multiprotein complex.The dynamic assembly of the CBM complex upon the engagement of AgR ultimately activates NF-κB and participates in the regulation of the immune processes. However the molecular mechanism relating to the assembly of CBM complex and the interaction among the three component all remain obscure.Our study investigated the mutual relation among the CBM base on the method and theory of protein structural biology. We have determined the crystal structure of the CARD domain from CARMA1(CARMA1-CARD) at high resolution, the structure consists of six helices as previously determined CARD domains. Furthermore, structural and computational analysis identified the binding interface between CARMA1-CARD and BCL10-CARD, which involves a basic patch in CARMA1and an acidic region in BCL10. Site-directed mutation, co-immunoprecipitation and NF-κB activation assay confirmed that the interface is necessary for association and downstream signaling, several critical residues that contribute to the interaction between CARMA1and BCL10were identified. Our studies provide molecular insight into the assembly of CARMA1and BCL10. CRACR2A plays a important role in the process of SOCE(store-operated Ca2+entry). As a cytoplasmic Ca2+sensor, CRACR2A directly interact with STIM1and Orail in a Ca2+-sensitive way, and regulates Ca2+release-activated Ca2+(CRAC) channels mediated Ca2+entry. We explored the structure and function of CRACR2A through the method of protein crystallization in association with NMR.