Allosteric activation of MALT1 by its ubiquitin-binding Ig3 domain Rebekka Schairera,1, Gareth Hallb,c,1, Ming Zhanga,1,2, Richard Cowanb,c, Roberta Baravalleb,c, Frederick W. Muskettb,c, Peter J. Coombsd, Chido Mpamhangad, Lisa R. Haled, Barbara Saxtyd, Justyna Iwaszkiewicze, Chantal Décailleta, Mai Perrouda, Mark D. Carrb,c,3, and Margot Thomea,3 aDepartment of Biochemistry, University of Lausanne, 1066 Epalinges, Switzerland; bDepartment of Molecular and Cell Biology, University of Leicester, LE1 7RH Leicester, United Kingdom; cLeicester Institute of Structural and Chemical Biology, University of Leicester, LE1 7RH Leicester, United Kingdom; dLifeArc, Accelerator Building, Open Innovation Campus, SG1 2FX Stevenage, United Kingdom; and eSwiss Institute of Bioinformatics, 1015 Lausanne, Switzerland Edited by Tak W. Mak, University Health Network, Toronto, Canada, and approved December 30, 2019 (received for review July 23, 2019) The catalytic activity of the protease MALT1 is required for Using biochemical approaches, we have previously shown that adaptive immune responses and regulatory T (Treg)-cell develop- MALT1 activation requires its monoubiquitination on K644, a ment, while dysregulated MALT1 activity can lead to lymphoma. lysine residue situated at the surface of the Ig3 domain (15). MALT1 activation requires its monoubiquitination on lysine 644 Moreover, we demonstrated an interaction of ubiquitin with an (K644) within the Ig3 domain, localized adjacent to the protease unknown binding site within the C-terminal half of MALT1, which domain. The molecular requirements for MALT1 monoubiquitina- comprises the protease domain, the Ig3 domain, and a non- tion and the mechanism by which monoubiquitination activates structured C-terminal extension (15). However, the precise loca- MALT1 had remained elusive. Here, we show that the Ig3 domain tion of the ubiquitin-binding site and the way by which ubiquitin interacts directly with ubiquitin and that an intact Ig3-ubiquitin binding contributes to MALT1 activation have remained unknown. interaction surface is required for the conjugation of ubiquitin to Here, we present experimental evidence that suggests that K644. Moreover, by generating constitutively active MALT1 mutants ubiquitin binding to the Ig3 domain is required for MALT1 that overcome the need for monoubiquitination, we reveal an allosteric communication between the ubiquitination site K644, the monoubiquitination, which, in turn, perturbs an inhibitory in- Ig3-protease interaction surface, and the active site of the protease teraction of the Ig3 domain with the protease domain and in- domain. Finally, we show that MALT1 mutants that alter the Ig3- duces conformational changes within the catalytic domain that IMMUNOLOGY AND INFLAMMATION ubiquitin interface impact the biological response of T cells. Thus, lead to enhanced activity. ubiquitin binding by the Ig3 domain promotes MALT1 activation by Results an allosteric mechanism that is essential for its biological function. Monomeric Ubiquitin Binds to the Ig3 Domain of MALT1. We had protease | structure | ubiquitin | signaling | Tcell previously shown that ubiquitin can interact physically with a C- terminal portion of MALT1 that comprises its protease domain, he paracaspase MALT1 is a proteolytic enzyme whose the Ig3 domain, and a nonstructured C-terminal extension (15). Tfunction is essential for adaptive immune responses and the development of particular lymphocyte subsets, such as Treg cells, Significance marginal zone, and B1 B cells (1–6). MALT1 is activated upon triggering of the B- or T cell antigen receptors and other immu- The protease MALT1 has been shown to play an essential role in noreceptors, such as activating natural killer cell (NK) receptors or the adaptive immune response and the development of lym- Fc receptors (7). A variety of G protein coupled receptors and phoma. The catalytic activity of MALT1 is tightly regulated by certain tyrosine receptor kinases have also been reported to signal via monoubiquitination, however, how ubiquitin is conjugated to MALT1 (7). A common feature of MALT1-activating receptors is MALT1 and the manner by which this modification regulates their capacity to induce the formation of so-called CARMA-BCL10- MALT1 activity remains poorly understood. Our data suggest that MALT1 (CBM) complexes, comprising a CARD-containing scaffold the Ig3 domain of MALT1 physically recruits ubiquitin to enable protein, the adaptor protein BCL10, and MALT1 (8). monoubiquitination and that ubiquitin conjugation to MALT1 ac- Based on structural studies on in vitro reconstituted CBM tivates the protease by inducing conformational changes in the Ig3 signalosomes from lymphocytes, which contain the scaffold domain that are communicated to the active site. These findings protein CARMA1 (also known as CARD11), it has been pro- identify the Ig3 domain of MALT1 as a novel ubiquitin-binding posed that the CBM complex adopts a helical filamentous structure domain and provide insight into the molecular requirements for in which CARMA1 nucleates the polymerization of BCL10 into MALT1 activation that could be of therapeutic interest. filaments that can incorporate MALT1 (9, 10). MALT1 contains an N-terminal death domain (DD) that binds to the core of the BCL10 Author contributions: R.S., G.H., M.Z., R.C., R.B., F.W.M., P.J.C., C.M., L.R.H., B.S., M.D.C., and M.T. designed research; R.S., G.H., M.Z., C.D., and M.P. performed research; R.S., G.H., filament by interacting with the BCL10 CARD motif (9). The M.Z., R.C., R.B., F.W.M., P.J.C., C.M., L.R.H., B.S., J.I., M.D.C., and M.T. analyzed data; and MALT1 DD is followed by two Ig-like domains, the protease do- R.S., G.H., M.D.C., and M.T. wrote the paper. main, a third Ig-like domain, and an unstructured C-terminal ex- The authors declare no competing interest. tension (11, 12). The C-terminal part of MALT1 that comprises the This article is a PNAS Direct Submission. protease domain is thought to protrude from the filamentous core Published under the PNAS license. (9), but the exact conformation and activation status of MALT1 1R.S., G.H., and M.Z. contributed equally to this work. within the BCL10/MALT1 fibers remain unknown. The crystallo- 2Present address: Division of Systems Biology and Personalised Medicine, The Walter and graphic analysis of highly purified constructs of MALT1 containing Eliza Hall Institute of Medical Research, 3052 Parkville, Melbourne, VIC, Australia. the protease and the adjacent Ig3 domain has revealed that the 3To whom correspondence may be addressed. Email: [email protected] or Margot. protease domain can dimerize (13, 14). The dimer crystals addi- [email protected]. tionally show that the protease and Ig3 domains physically interact This article contains supporting information online at https://www.pnas.org/lookup/suppl/ and undergo a rotational movement upon binding of a substrate doi:10.1073/pnas.1912681117/-/DCSupplemental. analog (13, 14). www.pnas.org/cgi/doi/10.1073/pnas.1912681117 PNAS Latest Articles | 1of10 Downloaded by guest on September 27, 2021 To identify key ubiquitin residues involved in the binding to MALT1 residues involved in the interaction with ubiquitin, we MALT1, we monitored differences in the 15N/1H HSQC spec- then generated a soluble monomeric MALT1 construct com- trum of uniformly labeled ubiquitin (1–76) induced by the ad- prising the protease and Ig3 domain (residues 339–719) and dition of MALT1 (Fig. 1A). The observed minimal shifts in monitored changes in the 15N/1H TROSY spectrum of uniformly backbone amide signals of ubiquitin upon MALT1 addition labeled MALT1 induced by the addition of ubiquitin. Upon clearly identified a contiguous interaction surface showing sig- addition of increasing concentrations of free ubiquitin, we ob- nificant shifts for 10 residues, including I13, G47, K6, K48, H68, served significant shifts in the backbone amide signals of several Q49, L69, V70, L71, and R72, centered around I44 (Fig. 1A and residues of MALT1, including E624, I625, Y692, L695, E696, SI Appendix, Fig. S1A). These residues are predominantly lo- D697, and T698 (Fig. 1D and SI Appendix, Fig. S1B). These calized to the β3–β5 strands collectively forming a continuous residues are mainly located within the β3/β4 and β6/β7 loops at patch of ∼172 Å2 on the surface of ubiquitin (Fig. 1B). This the surface of the MALT1 Ig3 domain on the side opposing the surface patch comprises several positively charged amino acids, Ig3-protease domain interaction surface (Fig. 1E), which com- including K6, K48, H68, and R72, which together form a striking bine to form a negatively charged area of ∼246 Å2 on the surface positively charged region on ubiquitin (Fig. 1C). To identify the of MALT1 (Fig. 1F). To provide additional evidence for the A D K48 0.06 G47 E696 D697 0.40 Q49 L71 I13 0.20 0.04 V70 K6 L69 R72 0.10 H68 0.02 0.05 0.00 0.00 Chemical Shift Change (ppm) 010203040506070 Chemical Shift Change (ppm) 580 600 620 640 660 680 700 Residue Number Residue Number B C E F K6 K6 Ig3 Domain Protease H68 H68 E696 D697 K48 I44 K48 I44 180° Q49 Q49 R72 R72 0.0 0.1 -+ 0.0 0.2 -+ Chemical Shift Charge Chemical Shift Charge G H MALT1 alone 0.02 MALT1 + 1.6 mM Ubiquitin ] ] ] m m m p p p p p p [ [ [ 1 1 1 F F F 6 . 0 2 K702 1 4 . 5 1 8 1 . 8 4 . 2 0 1 2 1 6 . 5 1 E624 1 0 0 . 5 0.01 1 2 2 1 8 1 . 5 1 Ubiquitin WT 1 Y692 2 . 2 . 0 1 . 5 2 6 2 1 1 1 1 2 . 4 . 6 1 4 1 . 2 1 5 1 2 Ubiquitin K6A 1 4 MALT1 WT . 6 1 6 . 1 1 2 6 . 1 0.00 5 2 1 6 .