(MALT1) Paracaspase Region

Crystal structure of the mucosa-associated lymphoid tissue lymphoma translocation 1 (MALT1) paracaspase region Jong W. Yua,1,2, Philip D. Jeffreya, Jun Yong Haa, Xiaolu Yangb, and Yigong Shic,1 aDepartment of Molecular Biology, Lewis Thomas Laboratory, Princeton University, Princeton, NJ 08544; bDepartment of Cancer Biology and Abramson Family Cancer Research Institute, University of Pennsylvania School of Medicine, Philadelphia, PA 19104; and cCenter for Structural Biology, Center for Life Sciences, School of Life Sciences and School of Medicine, Tsinghua University, Beijing 100084, China Edited by Suzanne Cory, The Walter and Eliza Hall Institute, Melbourne, Victoria, Australia, and approved October 17, 2011 (received for review July 25, 2011) The mucosa-associated lymphoid tissue lymphoma translocation 1 Within the CBM signalosome, MALT1 is an essential compo- (MALT1) paracaspase, a key component of the Carma1/Bcl10/ nent and appears to transmit signals predominantly by recruiting MALT1 signalosome, is critical for NF-κB signaling in multiple con- and oligomerizing TRAF6 or other E3 ubiquitin ligases. In addi- texts. MALT1 is thought to function as a scaffold and protease to tion to its scaffolding function, MALT1 also possesses arginine- promote signaling; however, the biochemical and structural basis specific proteolytic activity that modulates NF-κB and possibly of paracaspase action remains largely unknown. Here we report other signaling pathways. For example, cleavage of the negative the 1.75-Å resolution crystal structure of the MALT1 paracaspase regulator A20 by MALT1 appears to disrupt the deubiquitylating region, which contains the paracaspase domain and an ensuing enzyme (DUB) activity of A20, leading to enhancement of NF-κB Ig-like domain. The paracaspase and the Ig domains appear as a signaling (10). Cleavage of Bcl10 by MALT1, on the other hand, single folding unit and interact with each other through extensive was reported to exert integrin-mediated cell adhesive properties van der Waals contacts and hydrogen bonds. The paracaspase do- independent of NF-κB (11). More recently, MALT1-mediated main adopts a fold that is nearly identical to that of classic caspases proteolysis of the DUB cylindromatosis (CYLD) has been impli- and homodimerizes similarly to form an active protease. Unlike cated in JNK activation during T-cell receptor signaling (12). BIOCHEMISTRY caspases, the active and mature form of the paracaspase domain Despite these tantalizing insights, the overall contribution of remains a single uncleaved polypeptide and specifically recognizes MALT1 protease activity to NF-κB and related pathways remains the bound peptide inhibitor Val-Arg-Pro-Arg. In particular, the car- largely enigmatic. Nevertheless, targeted inhibition of MALT1 boxyl-terminal amino acid Arg of the inhibitor is coordinated by proteolytic activity may contribute to the therapy of various dis- three highly conserved acidic residues. This structure serves as an eases where NF-κB and related pathways play a major role. In important framework for deciphering the function and mechanism support of this view, MALT1 protease inhibition has been shown of paracaspases exemplified by MALT1. to dramatically inhibit growth and promote apoptosis of the highly aggressive activated B cell-like form of diffuse large B cell he transcription factor NF-κB is a key constituent of all cell lymphomas (13, 14). types and is activated by various receptors to regulate survival, T Although MALT1 was initially linked to immune system func- proliferation, migration, and differentiation (1). In particular, tion, it has emerged as a critical component of NF-κB signaling in NF-κB functions early in the development and maintenance of multiple contexts. For example, MALT1 and Bcl10 have been innate and adaptive immune systems and execution of the im- shown to couple with different Carma1 homologues to transduce mune response. Although caspases, cysteine proteases that cleave κ substrate proteins after aspartate residues, are widely known as NF- B signaling downstream of multiple receptor types (15). It is executioners of programmed cell death or apoptosis (2), a subset unclear, however, how MALT1 integrates its scaffolding and pro- and related members also activate NF-κB to promote lymphocyte tease activities through its C-terminal region to modulate proper proliferation and inflammation. One such caspase-like family cellular responses in these different contexts. Sequence analysis member, the mucosa-associated lymphoid tissue lymphoma of MALT1 initially identified a region with loose homology to the κ translocation 1 (MALT1) paracaspase, was identified through caspase fold (3) followed by an Ig-like domain critical for NF- B weak sequence homology to caspases (3) and was subsequently activation (16). Subsequent investigations have failed to identify found to play an important role in lymphocyte activation (4) and significant similarity between MALT1 and classic caspases in terms disease progression in MALT lymphomas (5). of substrate recognition, activity and specificity, and autoproteoly- Upon antigen receptor stimulation, the MALT1 paracaspase tic maturation. Although the Ig domain had been implicated in and Bcl10 assemble into the Carma1/Bcl10/MALT1 (CBM) sig- modulating protease activity and recruiting components required nalosome to activate NF-κB in the adaptive immune system. Spe- for proper ubiquitin ligase activity (7, 16), the underlying mechan- cifically during T-cell receptor signaling, the CBM signalosome is ism remains unknown. thought to oligomerize MALT1 and its associated ubiquitin ligase tumor necrosis factor receptor-associated factor 6 (TRAF6) or Author contributions: J.W.Y. and Y.S. designed research; J.W.Y., P.D.J., and J.Y.H. performed TRAF2 (6, 7), which in turn facilitates K63-linked polyubiquity- research; J.W.Y. and X.Y. contributed new reagents/analytic tools; J.W.Y., P.D.J., J.Y.H., X.Y., lation of multiple proteins including the regulatory γ-subunit of and Y.S. analyzed data; and J.W.Y. and Y.S. wrote the paper. the IκB kinase (IKK) complex (6, 7), TRAF6 itself (7), Bcl10 (8), The authors declare no conflict of interest. and MALT1 (9). Poly ubiquitylation of these proteins ultimately This article is a PNAS Direct Submission. β leads to the recruitment of transforming growth factor -activated Data deposition: The atomic coordinates and structure factors have been deposited in the kinase 1 (TAK1), TAK1 binding protein (TAB), and the IKK Protein Data Bank, www.pdb.org (PDB ID codes 3UOA and 3UO8). β complex to lipid rafts where the IKK -subunit is phosphorylated 1To whom correspondence may be addressed. E-mail: [email protected] or shi-lab@ and activated. In the canonical NF-κB pathway, the activated IKK tsinghua.edu.cn. complex phosphorylates IκB, enabling proteasome-mediated de- 2Present address: GlaxoSmithKline, Collegeville, PA 19426. κ κ gradation of I B and subsequent translocation of NF- Bintothe This article contains supporting information online at www.pnas.org/lookup/suppl/ nucleus where it acts as a transcriptional activator. doi:10.1073/pnas.1111708108/-/DCSupplemental. www.pnas.org/cgi/doi/10.1073/pnas.1111708108 PNAS Early Edition ∣ 1of6 Downloaded by guest on September 26, 2021 To gain mechanistic insight into MALT1 function, we deter- molecule bound to a peptide inhibitor (Fig. 1B). Each MALT1-C mined the crystal structure of an inhibitor-bound MALT1 fragment protein adopts a cone-shaped structure, with the Ig fold as the containing the paracaspase domain and a critical Ig domain. Our sharp tip and the paracaspase domain as the blunt bottom of the study reveals a number of findings that may serve as a basis for cone. Together, the MALT1-C homodimer resembles a prolate understanding the mechanism of MALT1 function in the CBM spheroid, measuring 160 Å in length and 60 Å in diameter (Fig. 1B complex. and Fig. S2). Confirming previous conjecture, the paracaspase domain indeed adopts a caspase-like fold that closely resembles Results that of canonical caspases. The Ig fold packs against the β2 strand Crystallization and Structure Determination. Full-length human and the α1 and α2 helices of the caspase-like fold, resulting in the 2 MALT1 (residues 1–824) contains a death domain (DD) and two burial of approximately 1;348 Å surface area. The paracaspase- Ig domains at the N-terminal region, and a predicted caspase- Ig assembly appears to be a single folding unit glued together by a like domain (paracaspase domain) and Ig fold at the C-terminal combination of electrostatic and hydrophobic interactions (see half (Fig. 1A). The N-terminal region is thought to interact with below). The intervening loop between the paracaspase domain Bcl10 and assemble into the CBM signalosome, whereas the and Ig domain, which extends from strand β6 of the caspase-like C-terminal half is believed to harbor scaffolding and protease ac- fold to helix α1 of the Ig fold, is likely flexible in solution as it is tivities (4). We attempted to bacterially overexpress various partially disordered in the crystal structure (Fig. 1B). MALT1 fragments encompassing the C-terminal half. The para- Despite a relatively low sequence identity of 17% between the caspase domain in isolation (residues 339–561) was found to be MALT1 paracaspase domain and a representative caspase such extremely prone to aggregation and precipitation, suggesting that as caspase 9, the paracaspase fold is virtually identical to that of other domains might be required for stabilization

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