Structures of Autoinhibited and Polymerized Forms of CARD9 Reveal Mechanisms of CARD9 and CARD11 Activation
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ARTICLE https://doi.org/10.1038/s41467-019-10953-z OPEN Structures of autoinhibited and polymerized forms of CARD9 reveal mechanisms of CARD9 and CARD11 activation Michael J. Holliday 1, Axel Witt1, Alejandro Rodríguez Gama2, Benjamin T. Walters 3, Christopher P. Arthur4, Randal Halfmann2,5, Alexis Rohou 4, Erin C. Dueber1 & Wayne J. Fairbrother 1 1234567890():,; 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, illumi- nating 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. 1 Early Discovery Biochemistry Department, Genentech, South San Francisco, CA 94080, USA. 2 Stowers Institute for Medical Research, Kansas City, MO 64110, USA. 3 Biochemical and Cellular Pharmacology Department, Genentech, South San Francisco, CA 94080, USA. 4 Structural Biology Department, Genentech, South San Francisco, CA 94080, USA. 5 Department of Molecular and Integrative Physiology, University of Kansas Medical Center, Kansas City, KS 66160, USA. Correspondence and requests for materials should be addressed to A.R. (email: [email protected]) or to E.C.D. (email: [email protected]) or to W.J.F. (email: [email protected]) NATURE COMMUNICATIONS | (2019) 10:3070 | https://doi.org/10.1038/s41467-019-10953-z | www.nature.com/naturecommunications 1 ARTICLE NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-019-10953-z aspase recruitment domain (CARD)-containing protein 9 shown to exhibit constitutive CARD11 signaling and aberrant cell C(CARD9) and CARD-containing protein 11 (CARD11, proliferation, confirming CARD11 as an oncogene27–29. Among aka. CARMA1) are paralogous proteins that act in a these studies, the N-terminal CARD and coiled-coil domains conserved manner as scaffolding proteins required to propagate emerged as mutational “hot-spots”, leading Chan et al.30 to signaling in immune cells1. CARD9 functions in myeloid cells conduct a high-throughput mutagenesis screen on the N-terminal during innate immune responses to activate NF-κB and p38 140 residues of CARD11, in the context of the full-length protein, MAPK. The most well characterized triggers of CARD9 signaling in search of hyperactivating CARD11 mutants. This study iden- are fungal carbohydrates, which interact with C-type lectin tified 23 sites with hyperactivating mutations in CARD11, many receptors and ultimately contribute to a Th17-type immune of which mapped to a short stretch of residues (112–130) termed response critical for proper control of fungal infections2–5. the “LATCH“, that was shown to be critical in maintaining CARD11 acts in lymphoid cells downstream of T- and B-cell CARD11 in an autoinhibited state. receptors and also leads to activation of NF-κB6,7. In these cell Here, we present a structure of the N-terminal region of types, CARD11 is critical for proper cellular activation and pro- CARD9, which exhibits an extensive autoinhibitory interface liferation upon receptor engagement. required to prevent constitutive activation in both CARD9 and CARD9 and CARD11 share an N-terminal domain archi- CARD11. From this structural insight, we then define the distinct tecture comprising an N-terminal CARD followed by ~300 structural mechanisms of activation in CARD9 and CARD11 and residues with high coiled-coil propensity, referred to as the demonstrate that, upon activation, both proteins form helical coiled-coil domain. CARD11 additionally contains a C-terminal templates that directly nucleate Bcl10 polymerization. Finally, membrane-associated guanylate kinase (MAGUK) domain, as solving a Cryo-EM structure of the CARD9 CARD helical well as a linker domain between the coiled-coil and MAGUK assembly, we structurally characterize the activated form of this domains known as the inhibitory domain (ID). Both CARD9 and protein family. CARD11 are thought to be held in autoinhibited states prior to activation, although the specific nature of these states remained uncharacterized prior to this study8. CARD11 is activated via Results phosphorylation of serine residues in the ID by protein kinase C, An extensive CARD-coiled-coil interface in CARD9. Both which interferes with a complex network of interactions between CARD9 and CARD11 are thought to be maintained in an auto- the ID, CARD, and coiled-coil domains required to maintain the inhibited state, but the structural basis for this inhibition had autoinhibited state9,10. CARD9 activation has been reported by remained an open question. To probe the mechanism of inhibi- different groups to require either phosphorylation at T231 by tion, we expressed and purified 15N-labeled regions of both PKCδ11 or ubiquitination at Lys125 by TRIM6212. To our CARD9 and CARD11 comprising the CARD and the first pre- knowledge, no single study has investigated both of these mod- dicted segment of coiled-coil that mediates homo-dimerization of ifications together, and, absent structural characterization of the each construct (CARD92–152 and CARD118–172, see Fig. 1a). system, no mechanistic explanation for CARD9 activation by Notably, CARD118–172 has been shown by Qiao et al.15 to be either of them has been suggested. sufficient to accelerate Bcl10 polymerization in vitro. While Upon activation, both CARD9 and CARD11 recruit the same CARD118–172 remained stable and soluble at high concentrations, downstream binding partner, B-cell lymphoma/leukemia 10 the construct exhibited a “molten globule” 15N-TROSY spectrum (Bcl10), which interacts through its own CARD with the CARD (Fig. 1b), indicative of significant conformational heterogeneity of CARD9 or CARD11, a CARD–CARD interaction critical for and/or self-association. While SEC-MALS analysis of subsequent signal propagation and NF-κB activation. The CARDs CARD118–172 indicated a molecular weight of 36.5 kDa, con- of CARD9 or CARD11 are thought to form a nucleating helical sistent with an expected dimeric molecular weight of 39.4 kDa, template that promotes polymerization of Bcl10 via the Bcl10 dynamic light scattering analysis at 100 μM revealed a poly- CARD, which, along with other domains of activated CARD9/11, dispersity index of 0.40, indicating some self-association, which then recruits downstream factors, including MALT1, cIAPs, was corroborated by a slight improvement in the nuclear mag- TRAF6, and the LUBAC complex that mediate subsequent ubi- netic resonance (NMR) spectral quality upon twofold dilution quitination of multiple members of the complex, including both (Supplementary Fig. 1A). In contrast to CARD118–172, the linear and K63-linked poly-ubiquitination of Bcl1013–16. These CARD11 CARD alone exhibits a well-dispersed spectrum indi- ubiquitination events ultimately lead to activation of IΚΚ and cative of a single dominant conformational state31. subsequent phosphorylation, ubiquitination, and degradation of Unlike CARD118–172, the homologous CARD92–152 construct IκB, thereby releasing NF-κB to translocate to the nucleus and (see Supplementary Fig. 1B for sequence alignment) exhibited a induce transcription. Regulation of the pathway at CARD9/11 is well-dispersed 15N-TROSY spectrum with fewer than 150 peaks thus likely achieved by modulating assembly of the helical CARD (Fig. 1c, black), consistent with a single dominant conformation template. and minimal self-association. We found that, at the high A number of disease-associated mutations have been identified concentrations required to collect three-dimensional (3D) NMR in CARD9 and CARD11. In CARD9, genetic deletion in mice or spectra for chemical shift assignment and structure determination loss-of-function mutations in humans lead to chronic fungal efforts, CARD92–152 formed higher-order oligomers over the infections17–21. One intriguing CARD9 splice variant, identified course of several hours, severely impacting NMR spectral quality as protective in inflammatory bowel disease, eliminates a TRIM62 (Supplementary Fig. 1C). We have shown previously that the binding site, preventing CARD9 signaling and bolstering the idea CARD9 CARD is capable of forming an extensively domain- that TRIM62 ubiquitination of CARD9 is critical for its swapped dimer, and suspected that these oligomers were due to 12,22 “ ” activation . In CARD11, genetic deletion in mice or loss-of- swap-dimer-mediated daisy-chaining of two or more coiled- function mutations in humans lead to immunodeficiency due to coil-mediated dimers31.Zn2+ binds to the CARD with sub- defects in activation and proliferation of both T- and B-cells23,24. nanomolar affinity and slows interconversion of the monomer A large number of mutations have also been identified in and domain-swapped CARD dimer ~50-fold, so we generated CARD11 associated with a range of lymphoproliferative dis-