Structural delineation and phase-dependent activation of the costimulatory CD27:CD70 complex

CD27 is a tumor necrosis factor (TNF) receptor, which stimulates lymphocytes and promotes their differentiation upon activation by TNF ligand CD70. Activation of the CD27 receptor provides a costimulatory signal to promote T cell, B cell, and NK cell activity to facilitate antitumor and anti-infection immunity. Aberrant increased and focused expression of CD70 on many tumor cells renders CD70 an attractive therapeutic target for direct tumor killing. However, despite their use as drug targets to treat cancers, the molecular basis and atomic details of CD27 and CD70 interaction remain elusive. Here we report the crystal structure of human CD27 in complex with human CD70. Analysis of our structure shows that CD70 adopts a classical TNF ligand homotrimeric assembly to engage CD27 receptors in a 3:3 stoichiometry. By combining structural and rational mutagenesis data with reported disease-correlated mutations, we identified the key amino acid residues of CD27 and CD70 that control this interaction. We also report increased potency for plate-bound CD70 constructs compared with solution-phase ligand in a functional activity to stimulate T-cells in vitro. These findings offer new mechanistic insight into this critical costimulatory interaction. CD27 is a tumor necrosis factor (TNF) receptor, which stimulates lymphocytes and promotes their differentiation upon activation by TNF ligand CD70. Activation of the CD27 receptor provides a costimulatory signal to promote T cell, B cell, and NK cell activity to facilitate antitumor and anti-infection immunity. Aberrant increased and focused expression of CD70 on many tumor cells renders CD70 an attractive therapeutic target for direct tumor killing. However, despite their use as drug targets to treat cancers, the molecular basis and atomic details of CD27 and CD70 interaction remain elusive. Here we report the crystal structure of human CD27 in complex with human CD70. Analysis of our structure shows that CD70 adopts a classical TNF ligand homotrimeric assembly to engage CD27 receptors in a 3:3 stoichiometry. By combining structural and rational mutagenesis data with reported disease-correlated mutations, we identified the key amino acid residues of CD27 and CD70 that control this interaction. We also report increased potency for plate-bound CD70 constructs compared with solution-phase ligand in a functional activity to stimulate T-cells in vitro. These findings offer new mechanistic insight into this critical costimulatory interaction. CD27 and CD70 are a receptor:ligand pair whose signaling activates and differentiates both T cells and B cells (1Han B.K. Olsen N.J. Bottaro A. The CD27-CD70 pathway and pathogenesis of autoimmune disease.Semin. Arthritis Rheum. 2016; 45: 496-501Crossref PubMed Scopus (34) Google Scholar). CD27 is a receptor belonging to the TNF (tumor necrosis factor) receptor superfamily. CD27 expression has been observed in a majority of peripheral blood T cells with high CD27 surface expression detected on activated T cells (2Hintzen R. De Jong R. Lens S. Brouwer M. Baars P. Van Lier R. Regulation of CD27 expression on subsets of mature T-lymphocytes.J. Immunol. 1993; 151: 2426-2435PubMed Google Scholar, 3Buchan S.L. Rogel A. Al-Shamkhani A. The immunobiology of CD27 and OX40 and their potential as targets for cancer immunotherapy.Blood. 2018; 131: 39-48Crossref PubMed Scopus (117) Google Scholar, 4Thiemann M. Richards D.M. Heinonen K. Kluge M. Marschall V. Merz C. Redondo Müller M. Schnyder T. Sefrin J.P. Sykora J. Fricke H. Gieffers C. Hill O. A single-chain-based hexavalent Cd27 agonist enhances T cell activation and induces anti-tumor immunity.Front. Oncol. 2018; 8: 387Crossref PubMed Scopus (7) Google Scholar). CD27 is also expressed on other lymphocytes including germinal center B cells, memory B cells, plasma B cells, and NK cells (3Buchan S.L. Rogel A. Al-Shamkhani A. The immunobiology of CD27 and OX40 and their potential as targets for cancer immunotherapy.Blood. 2018; 131: 39-48Crossref PubMed Scopus (117) Google Scholar). The receptor CD27 can be shed from the cell surface through the activity of MMP-8 and the resulting soluble fragment has been shown to inhibit signaling, likely due to competition with intact CD27 for ligand (5Kato K. Chu P. Takahashi S. Hamada H. Kipps T.J. Metalloprotease inhibitors block release of soluble CD27 and enhance the immune stimulatory activity of chronic lymphocytic leukemia cells.Exp. Hematol. 2007; 35: 434-442Abstract Full Text Full Text PDF PubMed Scopus (24) Google Scholar, 6Zhou Y. Liu X. Xu L. Tseng H. Cao Y. Jiang J. Ciccarelli B.T. Yang G. Patterson C.J. Hunter Z.R. Treon S.P. Matrix metalloproteinase-8 is overexpressed in Waldenstrom's macroglobulinemia cells, and specific inhibition of this metalloproteinase blocks release of soluble CD27.Clin. Lymphoma Myeloma Leuk. 2011; 11: 172-175Abstract Full Text Full Text PDF PubMed Scopus (12) Google Scholar). The TNF ligand superfamily member CD70 is the only known ligand of CD27. CD70 exhibits transient surface expression on macrophages and mature DCs upon cell activation (7Denoeud J. Moser M. Role of CD27/CD70 pathway of activation in immunity and tolerance.J. Leukoc. Biol. 2011; 89: 195-203Crossref PubMed Scopus (122) Google Scholar). CD70 expression is also detected on NK cells, B cells, and activated T cells (1Han B.K. Olsen N.J. Bottaro A. The CD27-CD70 pathway and pathogenesis of autoimmune disease.Semin. Arthritis Rheum. 2016; 45: 496-501Crossref PubMed Scopus (34) Google Scholar). The engagement of CD27 by CD70 induces signaling that affects many aspects of immune function. Downstream signaling depends on cytoplasmic segment of CD27 binding to TRAF (TNF receptor associated factor) signaling adaptors including TRAF2, TRAF3, and TRAF5 (8Yamamoto H. Kishimoto T. Minamoto S. NF-κB activation in CD27 signaling: Involvement of TNF receptor-associated factors in its signaling and identification of functional region of CD27.J. Immunol. 1998; 161: 4753-4759PubMed Google Scholar, 9Akiba H. Nakano H. Nishinaka S. Shindo M. Kobata T. Atsuta M. Morimoto C. Ware C.F. Malinin N.L. Wallach D. CD27, a member of the tumor necrosis factor receptor superfamily, activates NF-κB and stress-activated protein kinase/c-Jun N-terminal kinase via TRAF2, TRAF5, and NF-κB-inducing kinase.J. Biol. Chem. 1998; 273: 13353-13358Abstract Full Text Full Text PDF PubMed Scopus (206) Google Scholar). Both TRAF2 and TRAF5 can drive Janus kinase (JNK) activation, as well as canonical and alternative NF-κB activation, whereas TRAF3 inhibits the alternative NF-κB pathway (8Yamamoto H. Kishimoto T. Minamoto S. NF-κB activation in CD27 signaling: Involvement of TNF receptor-associated factors in its signaling and identification of functional region of CD27.J. Immunol. 1998; 161: 4753-4759PubMed Google Scholar, 9Akiba H. Nakano H. Nishinaka S. Shindo M. Kobata T. Atsuta M. Morimoto C. Ware C.F. Malinin N.L. Wallach D. CD27, a member of the tumor necrosis factor receptor superfamily, activates NF-κB and stress-activated protein kinase/c-Jun N-terminal kinase via TRAF2, TRAF5, and NF-κB-inducing kinase.J. Biol. Chem. 1998; 273: 13353-13358Abstract Full Text Full Text PDF PubMed Scopus (206) Google Scholar, 10Hauer J. Püschner S. Ramakrishnan P. Simon U. Bongers M. Federle C. Engelmann H. TNF receptor (TNFR)-associated factor (TRAF) 3 serves as an inhibitor of TRAF2/5-mediated activation of the noncanonical NF-κB pathway by TRAF-binding TNFRs.Proc. Natl. Acad. Sci. U. S. A. 2005; 102: 2874-2879Crossref PubMed Scopus (198) Google Scholar). In vitro activation of CD27 by CD70 coincident with TCR engagement stimulates both human αβ and γδ T cell lineages to proliferate, secrete cytokines, and prolong survival (3Buchan S.L. Rogel A. Al-Shamkhani A. The immunobiology of CD27 and OX40 and their potential as targets for cancer immunotherapy.Blood. 2018; 131: 39-48Crossref PubMed Scopus (117) Google Scholar). As an example of the importance of this costimulatory activity, patients with defective CD27:CD70 signaling caused by loss-of-function mutations are more susceptible to persistent EBV (Epstein–Barr virus) infections due to a paucity in memory B cells and EBV-specific T cells (both CD4+ and CD8+) (11Izawa K. Martin E. Soudais C. Bruneau J. Boutboul D. Rodriguez R. Lenoir C. Hislop A.D. Besson C. Touzot F. Picard C. Callebaut I. de Villartay J.P. Moshous D. Fischer A. et al.Inherited CD70 deficiency in humans reveals a critical role for the CD70–CD27 pathway in immunity to Epstein-Barr virus infection.J. Exp. Med. 2017; 214: 73-89Crossref PubMed Scopus (82) Google Scholar, 12Abolhassani H. Edwards E.S. Ikinciogullari A. Jing H. Borte S. Buggert M. Du L. Matsuda-Lennikov M. Romano R. Caridha R. Combined immunodeficiency and Epstein-Barr virus–induced B cell malignancy in humans with inherited CD70 deficiency.J. Exp. Med. 2017; 214: 91-106Crossref PubMed Scopus (102) Google Scholar). These patients are consequently more likely to present with recurrent EBV-driven lymphoproliferative disorder and EBV-positive lymphoma (11Izawa K. Martin E. Soudais C. Bruneau J. Boutboul D. Rodriguez R. Lenoir C. Hislop A.D. Besson C. Touzot F. Picard C. Callebaut I. de Villartay J.P. Moshous D. 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The CD27:CD70 pathway does not only affect T cell activity as B cell activation and NK cell survival are also induced via reverse CD70 signaling upon CD27 receptor binding (3Buchan S.L. Rogel A. Al-Shamkhani A. The immunobiology of CD27 and OX40 and their potential as targets for cancer immunotherapy.Blood. 2018; 131: 39-48Crossref PubMed Scopus (117) Google Scholar). CD70-mediated reverse signaling, which acts through PI3K and MEK transduction pathways (14Jacobs J. Zwaenepoel K. Rolfo C. Van den Bossche J. Deben C. Silence K. Hermans C. Smits E. Van Schil P. Lardon F. Deschoolmeester V. Pauwels P. Unlocking the potential of CD70 as a novel immunotherapeutic target for non-small cell lung cancer.Oncotarget. 2015; 6: 13462-13475Crossref PubMed Scopus (30) Google Scholar), may explain the role of CD70 in tumor biology. CD70 expression is limited in normal tissues but is strongly expressed in many lymphomas and solid tumors including pancreatic, lung, renal and ovarian cancers (15Aftimos P. Rolfo C. Rottey S. Offner F. Bron D. Maerevoet M. Soria J.-C. Moshir M. Dreier T. Van Rompaey L. Phase I dose-escalation study of the anti-CD70 antibody ARGX-110 in advanced malignancies.Clin. Cancer Res. 2017; 23: 6411-6420Crossref PubMed Scopus (27) Google Scholar, 16Wajant H. Therapeutic targeting of CD70 and CD27.Expert Opin. Ther. Targets. 2016; 20: 959-973Crossref PubMed Scopus (34) Google Scholar). The aberrant increase in CD70 expression on tumor cells correlates with increased proliferation and tumor survival (17Lens S.M. Drillenburg P. Den Drijver B.F. Van Schijndel G. Pals S.T. Van Lier R.A. Van Oers M.H. Aberrant expression and reverse signalling of CD70 on malignant B cells.Br. J. Haematol. 1999; 106: 491-503Crossref PubMed Scopus (100) Google Scholar, 18Riether C. Schürch C.M. Bührer E.D. Hinterbrandner M. Huguenin A.-L. Hoepner S. Zlobec I. Pabst T. Radpour R. Ochsenbein A.F. CD70/CD27 signaling promotes blast stemness and is a viable therapeutic target in acute myeloid leukemia.J. Exp. Med. 2017; 214: 359-380Crossref PubMed Scopus (67) Google Scholar). Mouse models have shown that chronic CD70 expression promotes CD8+ T cell exhaustion and impairs CD8+ T cell differentiation, while clinical studies demonstrate that CD70 blockade decreases Treg frequency and activity (15Aftimos P. Rolfo C. Rottey S. Offner F. Bron D. Maerevoet M. Soria J.-C. Moshir M. Dreier T. Van Rompaey L. Phase I dose-escalation study of the anti-CD70 antibody ARGX-110 in advanced malignancies.Clin. Cancer Res. 2017; 23: 6411-6420Crossref PubMed Scopus (27) Google Scholar, 19van Gisbergen K.P. van Olffen R.W. van Beek J. van der Sluijs K.F. Arens R. Nolte M.A. van Lier R.A. Protective CD8 T cell memory is impaired during chronic CD70-driven costimulation.J. Immunol. 2009; 182: 5352-5362Crossref PubMed Scopus (34) Google Scholar, 20Tesselaar K. Arens R. van Schijndel G.M. Baars P.A. van Der Valk M.A. Borst J. van Oers M.H. van Lier R.A. Lethal T cell immunodeficiency induced by chronic costimulation via CD27-CD70 interactions.Nat. Immunol. 2003; 4: 49-54Crossref PubMed Scopus (176) Google Scholar). In response to these observations, multiple antibodies and antibody drug conjugates targeting tumor-associated CD70 have been developed to directly deplete tumor cells and reverse CD70-mediated immune suppression (3Buchan S.L. Rogel A. Al-Shamkhani A. The immunobiology of CD27 and OX40 and their potential as targets for cancer immunotherapy.Blood. 2018; 131: 39-48Crossref PubMed Scopus (117) Google Scholar). Due to the immunostimulatory function of the CD27-CD70 signaling axis, augmentation of CD27 signaling has been identified as a potential therapeutic approach to promote T cell activation and improve antitumor efficacy. Transgenic constitutive expression of CD70 on B cells or dendritic cells has been shown to spontaneously convert naïve T cells into effector cells, promote T cell memory, and elicit more robust cytotoxicity against tumor in transgenic mouse models (21Arens R. Schepers K. Nolte M.A. Van Oosterwijk M.F. Van Lier R.A. Schumacher T.N. Van Oers M.H. Tumor rejection induced by CD70-mediated quantitative and qualitative effects on effector CD8+ T cell formation.J. Exp. Med. 2004; 199: 1595-1605Crossref PubMed Scopus (125) Google Scholar, 22Keller A.M. Schildknecht A. Xiao Y. van den Broek M. Borst J. Expression of costimulatory ligand CD70 on steady-state dendritic cells breaks CD8+ T cell tolerance and permits effective immunity.Immunity. 2008; 29: 934-946Abstract Full Text Full Text PDF PubMed Scopus (119) Google Scholar). 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Merz C. Redondo Müller M. Schnyder T. Sefrin J.P. Sykora J. Fricke H. Gieffers C. Hill O. A single-chain-based hexavalent Cd27 agonist enhances T cell activation and induces anti-tumor immunity.Front. Oncol. 2018; 8: 387Crossref PubMed Scopus (7) Google Scholar, 24Buchan S.L. Fallatah M. Thirdborough S.M. Taraban V.Y. Rogel A. Thomas L.J. Penfold C.A. He L.-Z. Curran M.A. Keler T. PD-1 blockade and CD27 stimulation activate distinct transcriptional programs that synergize for CD8+ T-cell–driven antitumor immunity.Clin. Cancer Res. 2018; 24: 2383-2394Crossref PubMed Scopus (51) Google Scholar). As a result of efficacy in mouse models, agonistic antibodies targeting CD27 have been developed to treat cancer patients. Two different agonistic therapeutic antibodies (varlilumab and MK-5890) were reported to be well tolerated and exhibited clinical activity in patients with hematologic malignancies or solid tumors (25Burris H.A. Infante J.R. Ansell S.M. Nemunaitis J.J. Weiss G.R. 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Herein we report the first crystal structure of human CD27:CD70 complex and demonstrate that CD27 and CD70 interact with 3:3 stoichiometry, in a manner similar to many other TNF receptor and ligand assemblies. Our CD27:CD70 structure also reveals key determinants involved in receptor:ligand recognition and several unique structural features important for the selectivity of this system. Based on the structure of the CD27:CD70 complex, we designed and screened sets of CD27 and CD70 mutants to validate the key amino acids residues contributing to the binding interface. Finally, we also assessed the ability of recombinant CD70 constructs to activate T cells in vitro. The full ectodomain of human CD27 contains three CRDs (cysteine-rich domains) and a stalk region with one cysteine (C185), which mediates receptor dimerization in vivo through intermolecular disulfide bond formation (8Yamamoto H. Kishimoto T. Minamoto S. NF-κB activation in CD27 signaling: Involvement of TNF receptor-associated factors in its signaling and identification of functional region of CD27.J. Immunol. 1998; 161: 4753-4759PubMed Google Scholar, 28Camerini D. Walz G. Loenen W. Borst J. Seed B. The T cell activation antigen CD27 is a member of the nerve growth factor/tumor necrosis factor receptor gene family.J. Immunol. 1991; 147: 3165-3169PubMed Google Scholar). The human CD70 ectodomain contains a TNF homology domain and a stalk region. To minimize flexibility of CD27:CD70 protein complex for crystallization, we designed constructs encoding only a portion of the extracellular domains of these two binding partners (Fig. 1A and Fig. S1, A and B). To simplify the carbohydrate modification, we expressed both constructs in the presence of kifunensine and minimized the sugar modification by subsequent endoglycosidase (EndoH) digestion. Human CD27 protein could be readily deglycosylated with no significant aggregation (Fig. S2B). However, EndoH digestion of human CD70 protein resulted in aggregation (Fig. S2A). Therefore, we pursued structure determination using deglycosylated CD27 in complex with glycosylated CD70. SEC (size-exclusion chromatography) and CGE (capillary gel electrophoresis) indicated that deglycosylated CD27 and glycosylated CD70 were purified to homogeneity (Fig. 1B and Fig. S2, A and B). Molecular characterization by aSEC (analytical size-exclusion chromatography) suggested that CD70 forms a stable homotrimer and CD27 is a monomer in solution (Fig. 1C). To assess the interaction of purified CD27 and CD70, we prepared mixtures of purified constructs in a 1:1 monomer molar ratio and assayed for complexation by monitoring the appearance of higher-molecular-weight species by aSEC. When CD27 and CD70 proteins were combined and incubated, we observed peaks corresponding to each individual component, indicating that the affinity between CD27 and CD70 does not support formation of a stable complex in solution (Fig. 1C). We therefore quantified the binding affinity between CD27 and CD70 by SPR (surface plasmon resonance). CD70 binds to glycosylated and deglycosylated CD27 with a KD (equilibrium dissociation constant) of 134 ± 1 nM (average ka = 3.23 × 104 M−1s−1; kd = 4.33 × 10−3 s−1) and 118 ± 1 nM (average ka = 5.17 × 104 M−1s−1; kd = 6.08 × 10−3 s−1), respectively (Fig. 1, D and E). From the experimentally measured dissociation rate, the calculated half-life for these interactions is 2.67 and 1.90 min, respectively. This short half-life compared with the timescale of the SEC separation (more than 10 min) explains the lack of detectable complex by SEC. The binding affinities of glycosylated and deglycosylated CD27 to CD70 are similar, suggesting that the CD27 carbohydrate modifications do not significantly affect the interaction. Crystallization of a complex between CD27 and CD70 was achieved by combining the individually purified components at a 1:1 monomer molar ratio and concentrating the sample prior to crystallization screens. We obtained crystals that diffracted to 2.69 Å with a space group of I222 (Table 1). The crystallographic asymmetric unit contains a single copy of the CD27:CD70 assembly composed of three CD27 monomers binding to one centrally located noncrystallographic CD70 homotrimer (Fig. 2A and Fig. S2C). Each CD27 monomer engages along the groove formed by two adjacent CD70 monomers. The three CD27 molecules in the asymmetric unit align very closely, with aligned Cα RMSDs ranging between 0.19 and 0.22 Å. Comparison of the structures of CD27 with previously determined CD27:Fab (fragment antigen binding) structures (PDB entries: 5Tl5, 5TLJ, 5TLK) shows minimal structural deviations within individual CRD domains. Moderate inter-CRD motion was observed when aligning the structures of CD27 from the CD27:CD70 complex and the CD27:Fab complex from the PDB entry 5Tl5, with a CRD1-CRD2 hinge difference of 3.4 degrees and a CRD2-CRD3 hinge difference of 10.7 degrees (Fig. 2B) (29Teplyakov A. Obmolova G. Malia T.J. Gilliland G.L. Crystal structure of CD27 in complex with a neutralizing noncompeting antibody.Acta Crystallogr. F Struct. Biol. Commun. 2017; 73: 294-299Crossref PubMed Scopus (7) Google Scholar). One N-linked glycan distant from the CD70-binding interface is observed on N95 residue within CRD2 of CD27 (Fig. 2A and Fig. S2, C and H), which is consistent with our SPR results indicating that CD27 glycans do not contribute significantly to the interaction with CD70.Table 1Crystallographic data collection and refinement statisticsParameterhuman CD27:CD70 complexData collection Space groupI222 Unit cell a, b, c (Å)109.53, 113.63, 163.51 Unit cell α, β, γ (°)90 Resolution (Å)93.31–2.69 (2.98–2.69) No. of total reflections124,088 No. of unique reflections18,602 Rmerge (%)10.5 (118) Rpim (%)4.4 (47.9) Mean I/σ (I)12.0 (1.7) Spherical completeness (%)64.9 (12.4) Ellipsoidal completeness (%)91.1 (55.4) Multiplicity6.7 (7.0) CC1/20.998 (0.727) Wilson B-factor (Å2)54.4Refinement Resolution (Å)36.72–2.69 (2.85–2.69) No. of reflections18,587 Rfree test set reflections915 (4.92%) Rwork/Rfree (%)20.0/23.6 Mean B-factor (Å2)All atoms99.2Protein93.8Ligand193.9Water57.3 RMSDBond length (Å)0.008Bond angle (°)1.09PDB entry7KX0Values in parentheses correspond to the highest-resolution shell. Open table in a new tab Values in parentheses correspond to the highest-resolution shell. The CD70 homotrimer resembles the classical TNF ligand trimeric assembly. The biologically active CD70 is formed by three protomers, with each protomer adopting a "jelly-roll" fold built around two β sheets (Fig. 2, C and D). Comparison of the CD70 homotrimer with representative TNF ligand structures demonstrates that CD70 adopts a "blooming flower" trimeric arrangement similar to OX40L (Fig. S3C). Electron density corresponding to N-linked glycan modifications extends from CD70 residues N63 and N170 on each protomer (Fig. 2D and Fig. S2, C and E–G). A region of density extending from O6 of the proximal N-acetylglucosamine moiety on residue N63 is consistent with a core fucosylation modification (Fig. 2D and Fig. S2, E and F). Superimposing the structure of CD70 protomer with a classical TNF ligand FasL shows a similar structural organization with an RMSD of 1.08 Å on 64 aligned Cα (Fig. S2D). Alignment of the scaffold β strands of CD70 and FasL shows even closer structural similarity with an RMSD of 0.64 Å on 35 aligned Cα (Fig. S2D). The most prominent structural differences between CD70 and FasL are present in the connecting loops between β strands. A region of positive density along the CD70 trimer axis was identified. As mentioned above, the trimer is related by a threefold noncrystallographic symmetry; hence this additional density cannot be attributed to artifact of data processing. Based on the features of the density and the composition of the crystallization solution, a Tris molecule was modeled at this location (Fig. 2, E–G). This Tris molecule forms hydrogen bonds with each CD70 monomer, and it may be possible that the energetic stability of the trimer is strengthened by the contribution of these interactions. Further investigation into the effect of buffer components on CD70 homotrimer stability was warranted, and so differential scanning calorimetry (DSC) experiments were performed. The melting temperature of CD70 was found to increase when Tris was present, consistent with an energetic stabilization of the CD70 homotrimer upon binding of this small molecule (Fig. 2H). Interestingly, structures of TL1a (30Jin T. Guo F. Kim S. Howard A. Zhang Y.Z. X-ray crystal structure of TNF ligand family member TL1A at 2.1A.Biochem. Biophys. Res. Commun. 2007; 364: 1-6Crossref PubMed Scopus (33) Google Scholar) and mouse TNFα (31Baeyens K.J. De Bondt H.L. Raeymaekers A. Fiers W. De Ranter C.J. The structure of mouse tumour-necrosis factor at 1.4 A resolution: Towards modulation of its selectivity and trimerization.Acta Crystallogr. D Biol. Crystallogr. 1999; 55: 772-778Crossref PubMed Scopus (40) Google Scholar) also contain small molecules (glycerol, Tris, and isopropanol, respectively) bound along the ligand homotrimer axis. It may be of interest to assess activity or stability of these TNF family ligands in the presence and absence of these small molecules to ascertain the functional relevance, if any, of these hypothetical binding sites. As an example of the principle that ligand binding at the homotrimer axis may alter the stability of the macromolecular complex, a small-molecule SPD-304 was identified to bind a similar site in TNFα (32He M.M. Smith A.S. Oslob J.D. Flanagan W.M. Braisted A.C. Whitty A. Cancilla M.T. Wang J. Lugovskoy A.A. Yoburn J.C. Small-molecule inhibition of TNF-α.Science. 2005; 310: 1022-1025Crossref PubMed Scopus (339) Google Scholar). SPD-304 binds to preformed TNFα trimer and induces a subunit rotation that displaces one TNFα monomer from the complex (32He M.M. Smith A.S. Oslob J.D. Flanagan W.M. Braisted A.C. Whitty A. Cancilla M.T. Wang J. Lugovskoy A.A. Yoburn J.C. Small-molecule inhibition of TNF-α.Science. 2005; 310: 1022-1025Crossref PubMed Scopus (339) Google Scholar). Our structure provides a model that may be used to design a similar strategy to inhibit CD70 activity. There are two pairs of intramolecular disulfide bonds observed in the structure of CD70. One disulfide bond is formed between residues C115 and C151 located on CD loop and EF loop, respectively (Fig. 2D). This disulfide bond is conserved among classical TNF ligands such as TNFα, LIGHT, TL1A, and FasL, etc. (Fig. S3A). The other disulfide bond, involving C168 on G β strand and C133 located on the DE loop, is unique to CD70 and was not observed in the previously determined TNF ligand structures (Fig. 2D). This disulfide bond is unusual in its position, which lies on the surface of the CD70 polypeptide (Fig. 2D) with 76.9% of the total surface area of the two side chain sulfur atoms exposed to solvent, except for the involvement of an additional posttranslational modification. This disulfide bond is shielded by the N-linked glycan extending from N170, with the two N-acetylglucosamine moieties positioned directly over the disulfide bond (Fig. 2D). The disulfide-bonded cysteines and the observed shielding of N-linked glycosylation site are conserved in mouse CD70 (Fig. S3A), which is to be expected for structural features that are required for protein stability. Mutation of the glycosylation residues or the cysteines resulted in failure of protein expression and purification in Expi293 cells (data not shown). While the C133-C168 disulfide does not directly interact with CD27, it is likely that the conformational constraints imposed on the DE loop by the disulfide bond impact the CD70-CD27 interaction. Each CD27 molecule interacts with two CD70 monomers, forming an interface that buries a total of 2440 Å2 of solvent accessible surface area from both CD27 and CD70. Two adjacent CD70 protomers contribute similar surface areas. For example, 653 Å2 of surface area from CD27 chain F engages CD70 chain A and 707 Å2 interacts with CD70 chain B (Fig. 3, A and B). Domains CRD2 and CRD3 of CD27 dominate the CD70-binding interface, with only a single residue from CRD1, F48, engaged in a van der Waals interaction