An integrative protocol for the structure determination of the mouse ASC-PYD filament.

Several inflammasomes that activate as part of the eukaryotic innate immune response contain long helical filaments of the adaptor protein ASC as a central structural element. Here, we describe a technical protocol that has enabled the structure determination of the filament of the ASC pyrin domain. The protocol integrates data from cryo-electron microscopy and solid-state NMR spectroscopy into a single simulated annealing protocol to determine structural coordinates that fit all input data optimally. The structure shows that the ASC pyrin domain filament is formed by helical stacking of individual pyrin domains forms and that the CARD domains are flexibly attached to the filament outside. An artificial perturbation of the input data shows that the integrated structure determination protocol can allow high quality structures even at resolutions of the electron density map as low 8Å. The protocol is extendable to other structural input data from biochemical or biophysical experiments.

Coordinated regulation of scaffold opening and enzymatic activity during CARD11 signaling.

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.

Structures of autoinhibited and polymerized forms of CARD9 reveal mechanisms of CARD9 and CARD11 activation.

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.

The inflammasome adapter ASC assembles into filaments with integral participation of its two Death Domains, PYD and CARD.

The inflammasome is a multiprotein complex necessary for the onset of inflammation. The adapter protein ASC assembles inflammasome components by acting as a molecular glue between danger-signal sensors and procaspase-1. The assembly is mediated by ASC self-association and protein interactions via its two Death Domains, PYD and CARD. Truncated versions of ASC have been shown to form filaments, but information on the filaments formed by full-length ASC is needed to construct a meaningful model of inflammasome assembly. To gain insights into this system, we used a combination of transmission EM, NMR, and computational analysis to investigate intact ASC structures. We show that ASC forms ∼6-7-nm-wide filaments that stack laterally to form bundles. The structural characteristics and dimensions of the bundles indicate that both PYD and CARD are integral parts of the filament. A truncated version of ASC with only the CARD domain (ASCCARD) forms different filaments (∼3-4-nm width), providing further evidence that both domains work in concert in filament assembly. Ring-shaped protein particles bound to pre-existing filaments match the size of ASC dimer structures generated by NMR-based protein docking, suggesting that the ASC dimer could be a basic building block for filament formation. Solution NMR binding studies identified the protein surfaces involved in the ASCCARD-ASCCARD interaction. These data provide new insights into the structural underpinnings of the inflammasome and should inform future efforts to interrogate this important biological system.