RESUMEN
The activation of key signaling pathways downstream of antigen receptor engagement is critically required for normal lymphocyte activation during the adaptive immune response. CARD11 is a multidomain signaling scaffold protein required for antigen receptor signaling to NF-κB, c-Jun N-terminal kinase, and mTOR. Germline mutations in the CARD11 gene result in at least four types of primary immunodeficiency, and somatic CARD11 gain-of-function mutations drive constitutive NF-κB activity in diffuse large B cell lymphoma and other lymphoid cancers. In response to antigen receptor triggering, CARD11 transitions from a closed, inactive state to an open, active scaffold that recruits multiple signaling partners into a complex to relay downstream signaling. However, how this signal-induced CARD11 conversion occurs remains poorly understood. Here we investigate the role of Inducible Element 1 (IE1), a short regulatory element in the CARD11 Inhibitory Domain, in the CARD11 signaling cycle. We find that IE1 controls the signal-dependent Opening Step that makes CARD11 accessible to the binding of cofactors, including Bcl10, MALT1, and the HOIP catalytic subunit of the linear ubiquitin chain assembly complex. Surprisingly, we find that IE1 is also required at an independent step for the maximal activation of HOIP and MALT1 enzymatic activity after cofactor recruitment to CARD11. This role of IE1 reveals that there is an Enzymatic Activation Step in the CARD11 signaling cycle that is distinct from the Cofactor Association Step. Our results indicate that CARD11 has evolved to actively coordinate scaffold opening and the induction of enzymatic activity among recruited cofactors during antigen receptor signaling.
Asunto(s)
Inmunidad Adaptativa/genética , Proteínas Adaptadoras de Señalización CARD/química , Guanilato Ciclasa/química , Complejos Multiproteicos/química , Receptores de Antígenos/genética , Proteína 10 de la LLC-Linfoma de Células B/genética , Proteínas Adaptadoras de Señalización CARD/genética , Proteínas Adaptadoras de Señalización CARD/ultraestructura , Mutación de Línea Germinal/genética , Guanilato Ciclasa/genética , Guanilato Ciclasa/ultraestructura , Humanos , Proteínas Quinasas JNK Activadas por Mitógenos/genética , Células Jurkat , Activación de Linfocitos/genética , Proteína 1 de la Translocación del Linfoma del Tejido Linfático Asociado a Mucosas/genética , Complejos Multiproteicos/genética , Complejos Multiproteicos/ultraestructura , FN-kappa B/genética , Unión Proteica/genética , Conformación Proteica , Receptores de Antígenos/química , Transducción de Señal/genética , Serina-Treonina Quinasas TOR/genética , Ubiquitina-Proteína Ligasas/genéticaRESUMEN
CARD9 and CARD11 drive immune cell activation by nucleating Bcl10 polymerization, but are held in an autoinhibited state prior to stimulation. Here, we elucidate the structural basis for this autoinhibition by determining the structure of a region of CARD9 that includes an extensive interface between its caspase recruitment domain (CARD) and coiled-coil domain. We demonstrate, for both CARD9 and CARD11, that disruption of this interface leads to hyperactivation in cells and to the formation of Bcl10-templating filaments in vitro, illuminating the mechanism of action of numerous oncogenic mutations of CARD11. These structural insights enable us to characterize two similar, yet distinct, mechanisms by which autoinhibition is relieved in the course of canonical CARD9 or CARD11 activation. We also dissect the molecular determinants of helical template assembly by solving the structure of the CARD9 filament. Taken together, these findings delineate the structural mechanisms of inhibition and activation within this protein family.
Asunto(s)
Proteínas Adaptadoras de Señalización CARD/ultraestructura , Guanilato Ciclasa/ultraestructura , Dominios Proteicos , Proteína 10 de la LLC-Linfoma de Células B/metabolismo , Proteínas Adaptadoras de Señalización CARD/genética , Proteínas Adaptadoras de Señalización CARD/inmunología , Proteínas Adaptadoras de Señalización CARD/metabolismo , Microscopía por Crioelectrón , Guanilato Ciclasa/genética , Guanilato Ciclasa/inmunología , Guanilato Ciclasa/metabolismo , Células HEK293 , Humanos , Mutación , Resonancia Magnética Nuclear Biomolecular , Conformación Proteica en Hélice alfa , Multimerización de Proteína/inmunología , Proteínas Recombinantes/genética , Proteínas Recombinantes/inmunología , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/ultraestructura , Transducción de Señal/inmunologíaRESUMEN
Soluble guanylate cyclase (sGC) is the primary nitric oxide (NO) receptor in mammals and a central component of the NO-signaling pathway. The NO-signaling pathways mediate diverse physiological processes, including vasodilation, neurotransmission, and myocardial functions. sGC is a heterodimer assembled from two homologous subunits, each comprised of four domains. Although crystal structures of isolated domains have been reported, no structure is available for full-length sGC. We used single-particle electron microscopy to obtain the structure of the complete sGC heterodimer and determine its higher-order domain architecture. Overall, the protein is formed of two rigid modules: the catalytic dimer and the clustered Per/Art/Sim and heme-NO/O2-binding domains, connected by a parallel coiled coil at two hinge points. The quaternary assembly demonstrates a very high degree of flexibility. We captured hundreds of individual conformational snapshots of free sGC, NO-bound sGC, and guanosine-5'-[(α,ß)-methylene]triphosphate-bound sGC. The molecular architecture and pronounced flexibility observed provides a significant step forward in understanding the mechanism of NO signaling.