Mechanistic Basis for Functional Promiscuity in the TNF and TNF Receptor Superfamilies: Structure of the LIGHT:DcR3 Assembly

Liu, Weifeng; Zhan, Changhong; Cheng, Huiyong; Kumar, P. Rajesh; Bonanno, J.B.; Nathenson, Stanley G.; Almo, Steven C.

doi:10.1016/j.str.2014.06.013

Cited by 27 publications

(30 citation statements)

References 44 publications

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“…For the latter, a glycine-serine-linker of 10 amino acids and a single glycine were chosen to connect the ECDs in the scGITRL and scLIGHT, respectively. 22,23 SDS-PAGE analysis of the purified proteins revealed the corresponding band of the respective monomer (Fig. 6A) and size exclusion chromatography confirmed the presence of the monomer in a main peak (Fig.…”

Section: Resultsmentioning

confidence: 83%

Advancing targeted co-stimulation with antibody-fusion proteins by introducing TNF superfamily members in a single-chain format

et al. 2016

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Co-stimulation via receptors of the tumor necrosis factor superfamily (TNFSF) emerges as promising strategy to support antitumor immune responses. Targeted strategies with antibody-fusion proteins composed of a tumor-directed antibody part and the extracellular domain of a co-stimulatory ligand of the TNFSF constitute an attractive option to focus the co-stimulatory activity to the tumor site. Since TNFSF members intrinsically form functional units of non-covalently linked homotrimers, the protein engineering of suitable antibody-fusion proteins is challenging. Aiming for molecules of simple and stable configuration, we used TNFSF ligands in a single-chain format (scTNFSF), i.e., three units of the ectodomain connected by polypeptide linkers, folding into an intramolecular trimer. By fusing tumordirected scFv antibody fragments directed against EpCAM or FAP to co-stimulatory scTNFSF molecules (sc4-1BBL, scOX40L, scGITRL or scLIGHT), a set of monomeric scFv-scTNFSF fusion proteins was generated. In comparison to the scFv-TNFSF format, defined by intermolecular homotrimerization via the TNFSF part, scFv-scTNFSF showed equal or enhanced co-stimulatory activity despite reduced avidity in antibody binding. In addition, enhanced serum stability and improved bioavailability in mice were observed. We show that the scFv-scTNFSF format can be applied to various members of the TNFSF, presenting targeting-dependent co-stimulatory activity. Hence, this format exhibits favorable properties that make it a promising choice for further therapeutic fusion protein development.Abbreviations: EpCAM, epithelial cell adhesion molecule; ED-A, fibronectin extradomain A; FAP, fibroblast activation protein; GITRL, glucocorticoid-induced tumor necrosis factor receptor (GITR) ligand; LIGHT, homologous to lymphotoxins, shows inducible expression and competes with herpes simplex virus glycoprotein D for herpesvirus entry mediator (HVEM), a receptor expressed by T lymphocytes; mAb, monoclonal antibody; scFv, single-chain fragment variable; TNF, tumor necrosis factor; TNFSF, tumor necrosis factor superfamily; TRAIL, TNF-related apoptosis inducing ligand

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Section: Resultsmentioning

confidence: 83%

Advancing targeted co-stimulation with antibody-fusion proteins by introducing TNF superfamily members in a single-chain format

et al. 2016

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“…Such low conservation is unexpected at positions that are structurally or functionally important. Furthermore, a structure of the closest sequence homolog of 4-1BBL, LIGHT, published after the e4-1BBL structure, shows a canonical bell-shaped trimer structure (13). Thus, we questioned whether 4-1BBL might be capable of forming a canonical trimer structure in which case the previously reported structure could be inaccurate.…”

Section: Residues Flanking the Thd Of 4-1bbl Can Be Deleted/mutated Wmentioning

confidence: 95%

Crystal structure of the human 4-1BB/4-1BBL complex

Gilbreth

Oganesyan

Amdouni

et al. 2018

Journal of Biological Chemistry

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4-1BBL is a member of the tumor necrosis factor (TNF) superfamily and is the ligand for the TNFR superfamily receptor, 4-1BB. 4-1BB plays an immunomodulatory role in T cells and NK cells, and agonists of this receptor have garnered strong attention as potential immunotherapy agents. Broadly speaking, the structural features of TNF superfamily members, their receptors, and ligand-receptor complexes are similar. However, a published crystal structure of human 4-1BBL suggests that it may be unique in this regard, exhibiting a three-bladed propeller-like trimer assembly that is distinctly different from that observed in other family members. This unusual structure also suggests that the human 4-1BB/4-1BBL complex may be structurally unique within the TNF/TNFR superfamily, but to date no structural data have been reported. Here we report the crystal structure of the human 4-1BB/4-1BBL complex at 2.4-Å resolution. In this structure, 4-1BBL does not adopt the unusual trimer assembly previously reported, but instead forms a canonical bell-shaped trimer typical of other TNF superfamily members. The structure of 4-1BB is also largely canonical as is the 4-1BB/4-1BBL complex. Mutational data support the 4-1BBL structure reported here as being biologically relevant, suggesting that the previously reported structure is not. Together, the data presented here offer insight into structure/function relationships in the 4-1BB/4-1BBL system and improve our structural understanding of the TNF/TNFR superfamily more broadly.

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“…The corresponding receptor monomers bind on the outside at the interface between two ligand monomers ( Fig. 1) (17)(18)(19)(20)(21)(31)(32)(33)(34)(35)(36)(37)(38)(39)(40). This is critically important for receptor activation, as it ensures that only an intact trimeric ligand can trigger signaling because even the loss of a single ligand monomer results in the loss of two receptor-binding sites.…”

Section: A Trimeric Ligand Bound To Three Receptors Forms the Basic Umentioning

confidence: 99%

Structural principles of tumor necrosis factor superfamily signaling

2018

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The tumor necrosis factor (TNF) ligand and receptor superfamilies play an important role in cell proliferation, survival, and death. Stimulating or inhibiting TNF superfamily signaling pathways is expected to have therapeutic benefit for patients with various diseases, including cancer, autoimmunity, and infectious diseases. We review our current understanding of the structure and geometry of TNF superfamily ligands, receptors, and their interactions. A trimeric ligand and three receptors, each binding at the interface of two ligand monomers, form the basic unit of signaling. Clustering of multiple receptor subunits is necessary for efficient signaling. Current reports suggest that the receptors are prearranged on the cell surface in a "nonsignaling," resting state in a large hexagonal structure of antiparallel dimers. Receptor activation requires ligand binding, and cross-linking antibodies can stabilize the receptors, thereby maintaining the active, signaling state. On the other hand, an antagonist antibody that locks receptor arrangement in antiparallel dimers effectively blocks signaling. This model may aid the design of more effective TNF signaling-targeted therapies.

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Mechanistic Basis for Functional Promiscuity in the TNF and TNF Receptor Superfamilies: Structure of the LIGHT:DcR3 Assembly

Cited by 27 publications

References 44 publications

Advancing targeted co-stimulation with antibody-fusion proteins by introducing TNF superfamily members in a single-chain format

Advancing targeted co-stimulation with antibody-fusion proteins by introducing TNF superfamily members in a single-chain format

Crystal structure of the human 4-1BB/4-1BBL complex

Structural principles of tumor necrosis factor superfamily signaling

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