Structure of TIGIT Immunoreceptor Bound to Poliovirus Receptor Reveals a Cell–Cell Adhesion and Signaling Mechanism That Requires Cis-Trans Receptor Clustering
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Structure of TIGIT immunoreceptor bound to poliovirus receptor reveals a cell–cell adhesion and signaling mechanism that requires cis-trans receptor clustering Katharina F. Stengela,b, Kristin Harden-Bowlesc, Xin Yud, Lionel Rougea, Jianping Yina, Laëtitia Comps-Agrarc, Christian Wiesmanna,1, J. Fernando Bazana,b,2, Dan L. Eatonc, and Jane L. Grogand,3 aDepartment of Structural Biology, bDepartment of Early Discovery Biochemistry, cDepartment of Protein Chemistry, and dDepartment of Immunology Research, Genentech, Inc., South San Francisco, CA 94080 Edited by Lewis L. Lanier, University of California, San Francisco, CA, and approved February 14, 2012 (received for review December 14, 2011) Nectins (nectin1–4) and Necls [nectin-like (Necl1–5)] are Ig superfam- PVR, a prototypical Nectin/Necl family member, is notable ily cell adhesion molecules that regulate cell differentiation and tis- among the nectin/Necl family as it not only provides heterophilic sue morphogenesis. Adherens junction formation and subsequent interactions with other nectin family members, such as nectin-3 cell–cell signaling is initiated by the assembly of higher-order recep- (17, 18), but also it interacts with IgSF molecules on immune torclustersofcognatemoleculesonjuxtaposedcells.However, lymphocytes such as TIGIT, CD226 (also known as DNAM-1) the structural and mechanistic details of signaling cluster formation (19), and CD96 (20) to regulate immune responses (21). Liga- remain unclear. Here, we report the crystal structure of poliovirus re- tion of PVR induces tyrosine phosphorylation of the PVR ceptor (PVR)/Nectin-like-5/CD155) in complex with its cognate im- immunoreceptor tyrosine-based inhibitory motif (ITIM) domain munoreceptor ligand T-cell-Ig-and-ITIM-domain (TIGIT). The TIGIT/ and recruitment of Src kinases and SHP-2 (SH2-domain-con- PVR interface reveals a conserved specific “lock-and-key” interaction. taining tyrosine phosphatase-2) (2, 4, 22–24). Activation of PVR Notably, two TIGIT/PVR dimers assemble into a heterotetramer with with TIGIT has been shown to attenuate immune responses a core TIGIT/TIGIT cis-homodimer, each TIGIT molecule binding one in vivo, predominantly through activation and phosphorylation PVR molecule. Structure-guided mutations that disrupt the TIGIT/ of Erk and induction of the suppressive cytokine IL-10 from TIGIT interface limit both TIGIT/PVR-mediated cell adhesion and dendritic cells (4). Originally, PVR was classified as a nectin-like TIGIT-induced PVR phosphorylation in primary dendritic cells. Our molecule (Necl–5) largely on the basis of a shared intracellular cis-trans data suggest a receptor clustering mechanism for cell adhe- motif; however, sequence analysis suggests that PVR is more sion and signaling by the TIGIT/PVR complex and provide structural similar to the nectins (4). Recently, we identified PVR family insights into how the PVR family of immunoregulators function. signature sequences in the IgSF ectodomains of PVR, nectins, TIGIT, CD226, and CD96 (4). Despite being diverse in domain ectins (nectin1–4) and nectin-like (Necl1–5) molecules are architecture, all PVR family members share three unique and Nmembers of the large Ig superfamily (IgSF) of cell-surface highly conserved sequence motifs in the first immunoglobulin IMMUNOLOGY receptors that play central roles in cell adhesion, cell movement, variable (IgV) domain: the (V/I)(S/T)Q, AX6G, and T(F/Y)P proliferation, and survival and contribute to the morphogenesis motifs (4). Like other nectins, PVR can form homodimers and and differentiation of many cell and tissue types by inducing an multimers in cis on cells (1, 17). – intracellular signaling cascade (1 5). Nectins and Necls can Here we present the crystal structures of TIGIT alone and in function as both ligands and receptors and therefore are able to complex with PVR. The 2.9-Å resolution structure of TIGIT in signal bidirectionally into juxtaposed cells (3, 6). To mediate the complex with PVR reveals a distinct “lock-and-key” motif that is formation of cell adherens junctions, a model suggests that the highly conserved across the PVR family members and is critical for extracellular domains of these molecules form ligand-dependent the TIGIT–PVR binding. Notably, the structure revealed a heter- homo- or heterodimers in trans (between molecules located on the otetrameric assembly of two TIGIT molecules flanked by two PVR same or opposite cell surfaces, respectively) and lateral homo- molecules. We show that the core TIGIT/TIGIT interface is dis- dimers in cis, creating a tight network of nectin zippers between tinct from the PVR/TIGIT interface and can exist in preformed juxtaposed cells (7, 8). To date, structural and functional studies lateral cis-dimers at the cell surface. Disruption of these TIGIT suggest a mechanism whereby the cis-homodimerization of a re- dimers, by site-directed mutagenesis, impaired cell adhesion to ceptor on the same cell surface is followed by the formation of and signaling in PVR-expressing cell lines and primary human a trans-dimer between juxtaposed cells using identical protein dendritic cells (DCs). Our data show that the lateral TIGIT interfaces. This assembly is noteworthy because it requires a rear- homodimers, together with the trans-TIGIT/PVR heterodimers, rangement and breakup of the cis-homodimer followed by a trans- dimerization across the adherens junction. The cis-trans clustering is then initiated through another unknown protein interface, likely Author contributions: K.F.S., C.W., J.F.B., D.L.E., and J.L.G. designed research; K.F.S., K.H.-B., involving a different receptor domain. X.Y., L.R., J.Y., and L.C.-A. performed research; K.F.S., K.H.-B., X.Y., L.R., L.C.-A., C.W., J.F.B., Several high-affinity homophilic trans-interactions have been D.L.E., and J.L.G. analyzed data; and K.F.S., J.F.B., and J.L.G. wrote the paper. – described in detail for nectins/Necls and similar molecules (8 13). Conflict of interest statement: During these studies, all authors were employed by Gen- However, the structure and function of the presumably weaker entech, Inc., which develops and markets drugs for profit. lateral homophilic cis-dimers in cell adhesion and their role in This article is a PNAS Direct Submission. intracellular signaling is not known. Because all structures solved Data deposition: Crystallography, atomic coordinates, and structure factors reported in to date are homodimers, it is unclear if they represent the cis- this paper have been deposited in the Protein Data Bank, www.pdb.org (PDB ID codes or the trans-state. Thus, the question of how cis-trans hetero- 3UCR and 3UDW). dimerization drives cell adhesion and intracellular signaling 1Present address: Novartis, 4002 Basel, Switzerland. remains open and was the impetus for capturing the heterophilic 2Present address: NeuroScience, Osceola, WI, 54020. interaction of the poliovirus receptor (PVR; also known as CD155 3To whom correspondence should be addressed. E-mail: [email protected]. – fi or Necl 5) (14) with its high-af nity ligand TIGIT (T-cell-Ig-and- This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. ITIM domain) (4, 15, 16). 1073/pnas.1120606109/-/DCSupplemental. www.pnas.org/cgi/doi/10.1073/pnas.1120606109 PNAS | April 3, 2012 | vol. 109 | no. 14 | 5399–5404 Downloaded by guest on September 28, 2021 can oligomerize in a zipper-like fashion to facilitate adhesion of N-linked glycosylation sites of PVR (N105 and N120) and lo- adjacent immune cells and thereby form receptor clusters that are cated on each β-sheet of the IgV domain without interfering with required for effective activation of PVR signaling. TIGIT binding. TIGIT lacked carbohydrate modifications, as it was expressed in E. coli, but neither of the two potential glyco- Results sylation sites (TIGIT residues N32 and N101) is close to the Crystal Structures of TIGIT and TIGIT Bound to PVR. To understand PVR-binding site. Neither TIGIT nor PVR showed significant the molecular mechanism of the TIGIT/PVR trans-dimerization, conformational rearrangements upon complex formation we determined the crystal structures of TIGIT and TIGIT bound (TIGIT rmsd of 0.8 Å over 105 Cα residues, PVR RMSD of 1.88 to PVR. Our work and previous experiments have shown that the Å over 103 Cα atoms). The crystal structures confirm that, de- N-terminal IgV domain of human PVR (PVR D1) is important spite low sequence homology, both PVR and TIGIT IgV for TIGIT IgV binding (4). Human TIGIT IgV was expressed in domains are very similar to each other and so they are grouped Escherichia coli and purified from inclusion bodies. Similarly, in the PVR family of cell adhesion molecules (4). human PVR D1 domain was expressed in the insect cell-baculo- virus system, purified, and complexed with TIGIT IgV. This TIGIT/PVR Interface Contains Lock-and-Key Binding Pockets. In the complex was stable and showed that TIGIT IgV and PVR D1 are TIGIT/PVR trans-dimer complex structure, the TIGIT/PVR in- terface is formed by interactions between the front β-sheets (A′ necessary and sufficient for TIGIT/PVR complex formation (Fig. GFCC′C′′) of each molecule (Fig. 1 and Fig. S4A). Because re- S1). We crystallized TIGIT alone and TIGIT in complex with ceptor and ligand share the same IgV fold, the interface displays PVR and solved the structures at 2.7 and 2.9 Å resolution, re- approximate noncrystallographic twofold symmetry and is highly spectively (Table S1 and Fig. 1). complementary in shape and charge. Interestingly, the interface in The TIGIT IgV domain and PVR D1 have a typical and very β the TIGIT/PVR complex uses the same structural elements as similar Ig -sandwich fold (Fig. S2) that is also very similar to that other IgV homo- and heterodimers (Fig. S4 A–G). A similar in- of Necl-1 (Fig.