Dimerization of LTβRbyLTα1β2 is necessary and sufficient for signal transduction Jawahar Sudhamsua,1, JianPing Yina,1, Eugene Y. Chiangb, Melissa A. Starovasnika, Jane L. Groganb,2, and Sarah G. Hymowitza,2 Departments of aStructural Biology and bImmunology, Genentech, Inc., South San Francisco, CA 94080 Edited by K. Christopher Garcia, Stanford University, Stanford, CA, and approved October 24, 2013 (received for review June 6, 2013) Homotrimeric TNF superfamily ligands signal by inducing trimers survival in a xenogeneic human T-cell–dependent mouse model of of their cognate receptors. As a biologically active heterotrimer, graft-versus-host disease (GVHD) (11). Lymphotoxin(LT)α1β2 is unique in the TNF superfamily. How the TNFRSF members are typically activated by TNFSF-induced three unique potential receptor-binding interfaces in LTα1β2 trig- trimerization or higher order oligomerization, resulting in initiation ger signaling via LTβ Receptor (LTβR) resulting in lymphoid organ- of intracellular signaling processes including the canonical and ogenesis and propagation of inflammatory signals is poorly noncanonical NF-κB pathways (2, 3). Ligand–receptor interactions α β understood. Here we show that LT 1 2 possesses two binding induce higher order assemblies formed between adaptor motifs in sites for LTβR with distinct affinities and that dimerization of LTβR the cytoplasmic regions of the receptors such as death domains or α β fi by LT 1 2 is necessary and suf cient for signal transduction. The TRAF-binding motifs and downstream signaling components such α β β crystal structure of a complex formed by LT 1 2,LT R, and the fab as Fas-associated protein with death domain (FADD), TNFR1- fragment of an antibody that blocks LTβR activation reveals the associated protein with death domain (TRADD), and TNFR-as- lower affinity receptor-binding site. Mutations targeting each po- sociated factor (TRAF). In particular, LTβR signals via TRAF3 tential receptor-binding site in an engineered single-chain variant and the structure of a peptide derived from the intracellular region α β fi κ of LT 1 2 reveal the high-af nity site. NF- B reporter assays fur- of LTβR bound to the TRAF3 C-terminal domain revealed a 3:3 ther validate that disruption of receptor interactions at either site trimeric complex (12, 13). is sufficient to prevent signaling via LTβR. Most TNFSF ligands are compact homotrimers formed by IMMUNOLOGY protomers possessing a conserved beta-strand jellyroll fold. Each crystallography | cytokines | mechanism | biophysics protomer is formed by an inner and outer β-sheet consisting of strands A’AHCF and B’BGDE, respectively (14). In contrast, he TNF receptor and ligand superfamilies (TNFRSF and multidomain TNFRSFs have an elongated shape and are com- TTNFSF, respectively) play critical roles in mammalian bi- posed of pseudorepeats of ∼40 residue cysteine-rich domains ology and mediate proinflammatory immune responses. Lym- (CRDs). The extracellular domain (ECD) of LTβR comprises α β photoxin (LT)- and LT are two related TNFSF members four CRDs and is expected to have similar overall architecture to produced predominately by activated cells of the innate and other multidomain TNFRSF members such as TNFR1. Crystal α adaptive immune response. LT exists as a secreted homotri- structures of several ligand–receptor complexes in this super- α meric molecule (LT 3) that signals via TNFR1 and TNFR2, or family (15–23) revealed that receptors bind in a symmetrical as a heterotrimer with LTβ on the cell surface (major form LTα β , minor form LTα β ) and signals through the LTβ re- 1 2 2 1 Significance ceptor (LTβR) (1). As a heterotrimer rather than a homotrimer, LTα1β2 is unique in the TNFSF. The role of LT in the immune response has been well charac- Cytokines are proteins that modulate the activity of target cells terized as critical for the development and orchestration of robust via activation of cell-surface receptors. The trimeric cytokines immune responses (2). Signaling through LTβR, expressed on of the tumor necrosis factor superfamily typically signal by nonhematopoeitic cells and follicular dendritic cells, directs normal inducing homotrimerization of their cognate receptors. We use development of lymph nodes and appropriate germinal center ar- structural and biophysical approaches to show that the unique heterotrimeric tumor necrosis factor superfamily member Lym- chitecture via the elaboration of various cytokines and chemokines, α β as revealed in LTα-, LTβ-, or LTβR-deficient mice (3, 4). During photoxin (LT) 1 2 induces dimerization rather than trimerization fi of the LTβ Receptor (LTβR). Cellular signaling assays were used chronic immune responses, cellular effectors can in ltrate target β fi tissue and organize anatomically into de novo lymphoid structures, to show that dimerization of LT Rissuf cient to activate in- instigated and maintained by LT-mediated pathways (5). tracellular signaling processes. Furthermore, disruption of re- ceptor interactions at either site prevents signaling via LTβR, Surface LTα1β2 is detected on subsets of activated T and B cells and NK cells (6–9). Dysregulation of these immune cell challenging the existing paradigm that trimeric complexes are types underlies the pathogenesis of autoimmune diseases. In required for signal transduction by the TNF family cytokines. mouse models of rheumatoid arthritis (RA), experimental au- Author contributions: J.S., J.Y., E.Y.C., M.A.S., J.L.G., and S.G.H. designed research; J.S., toimmune encephalomyelitis (EAE), and delayed-type hyper- J.Y., E.Y.C., and S.G.H. performed research; J.S., E.Y.C., M.A.S., J.L.G., and S.G.H. analyzed sensitivity (DTH), treatment with a depleting antibody specificto data; and J.S., J.Y., E.Y.C., J.L.G., and S.G.H. wrote the paper. murine LTα resulted in amelioration of disease in all circum- Conflict of interest statement: All authors are employees of Genentech, Inc. fi stances. In these studies, the Fc-dependent ef cacy achieved with This article is a PNAS Direct Submission. α anti-LT resulted from depletion of pathogenic LT-expressing Freely available online through the PNAS open access option. β Th1 and Th17 cells. Moreover, blockade of LT R signaling using Data deposition: The atomic coordinates and structure factors have been deposited in the a decoy receptor fusion protein, LTβR-Ig, was sufficient to drive Protein Data Bank, www.pdb.org (PDB ID codes 4MXV and 4MXW). fi ef cacy in a number of autoimmune models when delivered 1J.S. and J.Y. contributed equally to this work. α preventatively (10). Motivated by the concerted effects of LT and 2To whom correspondence may be addressed. E-mail: [email protected] or grogan. LTβ in driving major inflammatory pathways and pathologies, we [email protected]. α previously generated a humanized anti-LT mAb (MLTA3698A, This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. hereafter referred to as anti-LTα), and demonstrated increased 1073/pnas.1310838110/-/DCSupplemental. www.pnas.org/cgi/doi/10.1073/pnas.1310838110 PNAS Early Edition | 1of6 Downloaded by guest on October 1, 2021 manner at the monomer–monomer interfaces of the ligands, This complex retains the threefold symmetry of the LTα3 trimer. with CRD2 and CRD3 mediating most receptor–ligand inter- Each LTα protomer binds one copy of anti-LTα Fab (Fig. 1D). actions (Fig. S1A). Strands A’,B’, and B and the DE-loop and the FG-loop on the Unlike most TNFSF ligands, biochemical studies surprisingly outer surface of the monomer comprise the majority of the anti- indicated that the LIGHT [TNFSF member homologous to LT, LTα epitope (Fig. 1E). This binding mode differs from that seen in inducible expression, competes with herpes simplex virus (HSV) a structure of CD40L bound to the fab fragment of a blocking glycoprotein D for HSV entry mediator (HVEM), a receptor antibody (24) in which the antibody directly competes for the re- expressed on T lymphocytes; TNFSF14] homotrimer and the ceptor-binding site. Like anti-LTα, the Fab fragment of infliximab LTα1β2 heterotrimer are only capable of binding two copies of with TNF (25) also binds to the outer surface of a single monomer their cognate receptor, LTβR. However, difficulty in making of TNF. However, the infliximab–TNF interaction centers on the recombinant, soluble LTα1β2 precluded further characterization EF loop in the tip of the trimer, whereas anti-LTα Fab binds to the (1). Nonetheless, the inherent asymmetry of the LTα1β2 hetero- wide part of the trimer. trimer suggests three distinct possible receptor-binding sites in Overlay of the LTα3–(anti–LTα-Fab)3 and the LTα3–TNFR13 LTα1β2 as opposed to three equivalent sites in a homotrimer (Fig. structures unexpectedly revealed minimal overlap between the S1 B and C). Herein we draw on structural, biochemical, and TNFR1 epitope and the anti-LTα epitope (Fig. S1 D and E). cellular data to show that dimeric clustering of LTβRbythe However, the Fab occupies part of the same 3D space as would LTα1β2 heterotrimer triggers signal transduction and that dis- be occupied by receptor during a signaling event, suggesting that rupting one receptor-binding site on LTα1β2 is sufficient to block steric hindrance contributes to the ability of anti-LTα to compete signaling through LTβR. with TNFR1 or TNFR2 for ligand. Anti-LTα binding also changes the conformation of the LTα DE and the AA’ loops, which con- Results tain the critical residues Y142 and D84, which are essential for Anti-LTα Binds to LTα at a Different Site from TNFR1 and Blocks receptor binding and cytotoxic activity of LTα (26). This confor- Signaling by Both TNFR1 and LTβR. To assess the binding of anti- mational change is an allosteric effect, as anti-LTα does not directly α α α β LT toward LT 3 and LT 1 2, we stimulated HeLa cells stably contact these residues.