Assembly mechanism of the CARMA1–BCL10–MALT1– TRAF6 signalosome Liron Davida,b, Yang Lia,b, Jun Mac, Ethan Garnerd, Xinzheng Zhangc, and Hao Wua,b,1 aDepartment of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, MA 02115; bProgram in Cellular and Molecular Medicine, Boston Children’s Hospital, Boston, MA 02115; cNational Laboratory of Biomacromolecules, Institute of Biophysics, Chinese Academy of Sciences, Beijing 100101, China; and dDepartment of Molecular and Cellular Biology, Center for Systems Biology, Harvard University, Cambridge, MA 02138 Contributed by Hao Wu, January 1, 2018 (sent for review December 19, 2017; reviewed by Andrew L. Snow and Jungsan Sohn) The CARMA1–BCL10–MALT1 (CBM) signalosome is a central medi- previously shown that CARMA1 nucleates BCL10 CARD fila- ator of T cell receptor and B cell receptor-induced NF-κB signaling ment formation (11), and these filaments may form the scaffold that regulates multiple lymphocyte functions. While caspase- for cytosolic clusters that are visible under light microscopy (12). recruitment domain (CARD) membrane-associated guanylate kinase MALT1 is a paracaspase with similarity to caspases, and its pro- (MAGUK) protein 1 (CARMA1) nucleates B cell lymphoma 10 (BCL10) teolytic activity is stimulated upon TCR engagement (13). Its filament formation through interactions between CARDs, mucosa- proteolytic activity is required for optimal NF-κB activation (13, associated lymphoid tissue lymphoma translocation protein 1 (MALT1) 14). TRAF6 is an adaptor protein with ubiquitin ligase activity that is a paracaspase with structural similarity to caspases, which recruits plays an important role in the signal transduction of TNF receptor A TNF receptor-associated factor 6 (TRAF6) for K63-linked polyubiquiti- superfamily, IL-1 receptors, and Toll-like receptors (Fig. 1 ). nation. Here we present cryo-electron microscopy (cryo-EM) struc- Upon recruitment to MALT1, TRAF6 mediates Lys63-linked polyubiquitination to elicit NF-κBactivation(15). ture of the BCL10 CARD filament at 4.0-Å resolution. The structure κ redefines CARD–CARD interactions compared with the previous EM TCR- and BCR-induced NF- B signaling has a crucial role in structure determined from a negatively stained sample. Surprisingly, regulating the activation, proliferation, and effector functions of time-lapse confocal imaging shows that BCL10 polymerizes in a uni- lymphocytes in adaptive immune responses (16, 17). Deregulation directional manner. CARMA1, the BCL10 nucleator, serves as a hub of this process results in immunodeficiency, autoimmune diseases, for formation of star-shaped filamentous networks of BCL10 and or neoplastic disorders. CARMA1 missense mutations were de- tected initially in human activated B cell-like (ABC)-diffuse large significantly decreases the lag period of BCL10 polymerization. Co- B cell lymphoma tumors (18) and more recently in B cell lym- operative MALT1 interaction with BCL10 filaments observed under phocytosis (19–21), adult T cell leukemia/lymphoma (22), and a EM suggests immediate dimerization of MALT1 in the BCL10 fila- form of cutaneous T cell lymphoma, named Sézary syndrome (23, mentous scaffold. In addition, TRAF6 cooperatively decorates CBM 24). Most mutations were mapped to within exons encoding the filaments to form higher-order assemblies, likely resulting in all-or- CARD, the coiled-coil domain, and the linker region, resulting in none activation of the downstream pathway. Collectively, these data overcoming autoinhibition and constitutive NF-κB activation. reveal biophysical mechanisms in the assembly of the CARMA1- MALT lymphoma is often associated with BCL10 and MALT1 BCL10-MALT1-TRAF6 complex for signal transduction. translocation-induced overexpression, as well as production of the CARMA1–BCL10–MALT1 | TRAF6 | innate immunity | cryo-EM | assembly Significance he ternary complex of caspase-recruitment domain (CARD) NF-κB family proteins are evolutionarily conserved master Tmembrane-associated guanylate kinase (MAGUK) protein 1 regulators of immune and inflammatory responses. They play (CARMA1, also known as CARD11), B cell lymphoma 10 (BCL10), critical roles in a wide array of biological processes, such as and mucosa-associated lymphoid tissue lymphoma translocation innate and adaptive immunity and embryonic development. BIOPHYSICS AND protein 1 (MALT1), known as the CBM complex, is indispensable – – COMPUTATIONAL BIOLOGY κ A The CARMA1 BCL10 MALT1 (CBM) complex is the central me- for NF- B signaling in both T and B lymphocytes (Fig. 1 ). In diator of T cell receptor and B cell receptor-induced NF-κBacti- T cells, upon stimulation by antigen peptides bound to MHC vation in lymphocytes. Mutations, chromosomal translocations, molecules on antigen-presenting cells, the T cell receptor (TCR) is θ θ and overexpression of CBM component proteins have shown to activated, which subsequently activates protein kinase C (PKC ). directly lead to non-Hodgkin’s lymphomas. Here we provide a In B cells, antigen stimulation of B cell receptor (BCR) leads to β θ comprehensive analysis of the CBM complex and its interaction activation and membrane recruitment of PKC .BothPKC and with TRAF6 using a combination of biochemical, structural, and PKCβ phosphorylate CARMA1 to induce conformational changes – imaging approaches to elucidate the molecular basis for the that activate CARMA1 (1 6). In the resting state, CARMA1 is assembly, activation, and disassembly. These data provide a supposed to exist in an autoinhibited conformation through intra- platform for the mechanistic understanding of associated lym- molecular interactions that involve multiple regions of the protein, phomas and potential therapeutic applications. including the CARD and the CC region (Fig. 1A). Phosphoryla- tion of CARMA1, mainly in the linker region, overcomes the intra- Author contributions: L.D. and H.W. designed research; L.D., Y.L., J.M., and X.Z. per- molecular interactions and releases the autoinhibition, although formed research; E.G. contributed new reagents/analytic tools; E.G. provided advice on details of the conformational change remain to be elucidated. Acti- imaging; L.D. and H.W. analyzed data; and L.D. and H.W. wrote the paper. vated CARMA1 induces the formation of the CARMA1–BCL10– Reviewers: A.L.S., Uniformed Services University of the Health Sciences; and J.S., The Johns MALT1 (CBM) complex and recruits TNF receptor-associated factor Hopkins University School of Medicine. 6 (TRAF6), resulting in NF-κBactivation. The authors declare no conflict of interest. CARMA1 contains a C-terminal MAGUK domain (7, 8) and Published under the PNAS license. belongs to the MAGUK family of proteins that function as mo- Data deposition: The atomic coordinates and cryo-EM map have been deposited in the lecular scaffolds to assist recruitment and assembly of signaling Protein Data Bank, www.rcsb.org (PDB ID code 6BZE) and EM Data Bank, emdatabank.org molecules at the cytoplasmic membrane (9). BCL10 contains an (ID code EMD-7314). N-terminal CARD and a C-terminal Ser/Thr-rich domain. While 1To whom correspondence should be addressed. Email: [email protected]. the CARD is involved in CARMA1 interaction, the C-terminal This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. region has been shown to interact with MALT1 (10). We have 1073/pnas.1721967115/-/DCSupplemental. www.pnas.org/cgi/doi/10.1073/pnas.1721967115 PNAS | February 13, 2018 | vol. 115 | no. 7 | 1499–1504 Fig. 1. Cryo-EM structure of BCL10 CARD filament. (A) Domain organization of CARMA1, BCL10, MALT1, and TRAF6. (B) A micrograph of negatively stained BCL10 (1–205) filaments. (C) Three-dimensional helical reconstruction (gray) of negatively stained BCL10 (1–205) filament, superimposed with fitted NMR structure of BCL10 CARD (cyan) (PDB ID code 2MB9). (D)Acryo-EM micrograph of BCL10 (1–205) filaments. (E) Cryo-EM 3D helical reconstruction (gray) of BCL10 (1–205) filament superimposed with refined atomic model (colored). (F) Ribbon representation of BCL10 subunit structure in the filament conformation based on the cryo-EM structure. (G) Schematic diagram of BCL10 CARD–CARD interaction types. (H)DetailedCARD–CARD interac- tions and their corresponding electrostatic surfaces. Type I interaction between two subunits is labeled in green and cyan, type II interaction between two sub- units is labeled in cyan, and type III interaction between two subunits is labeled in magenta and cyan. API2–MALT1 fusion protein that cause constitutive NF-κBacti- region in BCL10 that precedes the MALT1-binding site near the vation (25). Given the significance of the CBM–TRAF6 complex C terminus (Fig. 1A). in human physiology and diseases, it is important to understand its mechanism of assembly. Remodeled BCL10 CARD–CARD Interactions. Analysis of BCL10 cryo- EM structure supported our previous model obtained from negative- Results staining EM data (11) and also revealed additional insights into Cryo-Electron Microscopy Structure of BCL10 CARD at 4.0-Å Resolution. the CARD–CARD interactions due to changes in the fitted We searched for a construct of BCL10 that could produce straight subunit orientation. The following three types of CARD–CARD – and rigid filaments suitable for structure determination by cryo- interactions are present: type III interaction facilitates the charge EM. BCL10 CARD (residues 1–115) formed very long and straight charge intrastrand contact, whereas type I and type II interactions G filaments;