New insights on the interaction mechanism of rhTNFα with its antagonists Adalimumab and Etanercept (original) (raw)
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Crystal structure of TNF-alpha mutant R31D with greater affinity for receptor R1 compared with R2
1997
Crystal structures have been determined of recombinant human tumor necrosis factor-α (TNF-α) and its R31D mutant that preferentially binds to TNF receptor R1 with more than seven times the relative affinity of binding to receptor R2. Crystals of the wild-type TNF were of space group P4 1 2 1 2 and had unit cell dimensions of a ⍧ b ⍧ 94.7 and c ⍧ 117.4 Å. Refinement of the structure gave an Rfactor of 22.3% at 2.5 Å resolution. The crystals of TNF R31D mutant diffracted to 2.3 Å resolution, and were of identical space group to the wild type with unit cell dimensions of a ⍧ b ⍧ 95.4 and c ⍧ 116.2 Å, and the structure was refined to an R-factor of 21.8%. The trimer structures of the wild-type and mutant TNF were similar with a root mean square (r.m.s.) deviation of 0.56 Å for Cα atoms; however, the subunits within each trimer were more variable with an average r.m.s. deviation of 1.00 Å on Cα atoms for pairwise comparison of subunits. Model complexes of TNF with receptors R1 and R2 have been used to predict TNF-receptor interactions. Arg31 in all three subunits of wild-type TNF is predicted to form an ionic interaction with the equivalent glutamic acid in both receptors R1 and R2. Asp31 of the TNF R31D mutant is predicted to interact differently with the two receptors. The side chain of Asp31 in two subunits of the TNF mutant is predicted to form hydrogen bond interactions with Ser59 or Cys70 of R1, while it has no predicted interactions with R2. The loss of three strong ionic interactions of Arg31 and the electrostatic repulsion of Asp31 with Glu in the receptors is consistent with the reduced binding of the R31D mutant to both receptors relative to wild-type TNF. The replacement of these ionic interactions by two weaker hydrogen bond interactions between Asp31 of the R31D mutant and R1, compared with no interactions with R2, is in agreement with the observed preferential binding of the R31D mutant to R1 over R2. Analysis of the structure and function of receptor-discriminating mutants of TNF will help understand the biological role of TNF and facilitate its use as an antitumor agent.
Journal of Interferon & Cytokine Research, 1996
Different soluble human TNFR80 derivatives, a solubilized form of the complete TNFR80, the TNFR80 ex¬ tracellular domain, a secretory TNFR80 mutant (TR80TM-) with a deleted transmembrane region, and a TNFR80 immunoadhesin were produced in insect cells and characterized side by side with a recombinant hu¬ man TNFR60 extracellular domain with respect to TNF binding affinity and neutralization of TNF bioactivity. The construct TR80TM-and the solubilized complete TNFR80 revealed a similar TNF binding and neu¬ tralization capacity, which was superior to the monovalent TNFR80 extracellular domain and comparable to the bivalent TNFR80 immunoadhesin, already known as a potent TNF antagonist. Determination of ligand off rate constants of the various receptor constructs by surface plasmon resonance revealed a correlation of low off rates with a high TNF neutralization capacity. We propose that the high TNF binding and neutral¬ ization capacity of the solubilized complete TNFR80 and TR80TM~in comparison with the monovalent ex¬ tracellular TNR80 domain is due to a noncovalent self-aggregation of the receptors via their intracellular do¬ main. This finding suggests that efficient soluble TNF antagonists can be derived from TNFR themselves without the need of construction of TNFR Ig Fc fusion proteins.
Protein science : a publication of the Protein Society, 2017
Adalimumab and Infliximab are recombinant IgG1 monoclonal antibodies (mAbs) that bind and neutralize human tumor necrosis factor alpha (TNFα). TNFα forms a stable homotrimer with unique surface-exposed sites for Adalimumab, Infliximab and TNF receptor binding. Here, we report the structures of Adalimumab-TNFα and Infliximab-TNFα complexes modeled from negative stain EM and cryo-EM images. EM images reveal complex structures consisting of 1:1, 1:2, 2:2 and 3:2 complexes of Adalimumab-TNFα and Infliximab-TNFα. The 2:2 complex structures of Adalimumab-TNFα and Infliximab-TNFα show diamond-shaped profiles and the 2-D class averages reveal distinct orientations of the Fab domains, indicating different binding modes by Adalimumab and Infliximab to TNFα. After separation by size exclusion chromatography and analysis by negative stain EM, the 3:2 complexes of Adalimumab-TNFα or Infliximab-TNFα complexes are more complicated but retain features recognized in the 2:2 complexes. Preliminary cr...
Journal of Biological Chemistry, 2019
Etanercept is a soluble form of the tumor necrosis factor receptor 2 (TNFR2) that inhibits pathological tumor necrosis factor (TNF) responses in rheumatoid arthritis and other inflammatory diseases. However, besides TNF, etanercept also blocks lymphotoxin-(LT), which has no clear therapeutic value and might aggravate some of the adverse effects associated with etanercept. Poxviruses encode soluble TNFR2 homologs, termed viral TNF decoy receptors (vTNFRs), that display unique specificity properties. For instance, cytokine response modifier D (CrmD) inhibits mouse and human TNF and mouse LT, but it is inactive against human LT. Here, we analyzed the molecular basis of these immunomodulatory activities in the ectrome-lia virus-encoded CrmD. We found that the overall molecular mechanism to bind TNF and LT from mouse and human origin is fairly conserved in CrmD and dominated by a groove under its 50s loop. However, other ligand-specific binding determinants optimize CrmD for the inhibition of mouse ligands, especially mouse TNF. Moreover, we show that the inability of CrmD to inhibit human LT is caused by a Glu-Phe-Glu motif in its 90s loop. Importantly, transfer of this motif to etanercept diminished its anti-LT activity in >60-fold while weakening its TNF-inhibitory capacity in 3-fold. This new etanercept variant could potentially be used in the clinic as a safer alternative to conventional etanercept. This work is the most detailed study of the vTNFR-ligand interactions to date and illustrates that a better knowledge of vTNFRs can provide valuable information to improve current anti-TNF therapies.
Cytokine, 1995
Tumour Necrosis Factor (TNF) is a homotrimeric monokine originally described for its antitumour activity. Now it is known as one of the most pleiotropic cytokines mediating a broad spectrum of biological activities, such as cell proliferation, cytotoxicity, antiviral responses, and activation of transcription factors and cellular genes. TNF is considered a key regulatory molecule in immune and inflammatory reactions, and in a number of pathological conditions. 1-3 Two distinct, high-affinity TNF receptors of 55 kDa (TNF-R55) and 75 kDa (TNF-R75) have been identified and cloned. 4-7 Based on the characteristic, repeating, cysteine-rich motifs in the extracellular domain, both TNF-R55 and TNF-R75 belong to the nerve growth factor/TNF-R family. 8 The intracellular domains of the two TNF-Rs show no sequence homology, suggesting distinct biological functions. Although the signalling mechanism of the TNF-Rs is still incompletely understood, oligomerization of receptors is believed to be a first essential step. This is supported by the fact that TNF activities can be mimicked with specific anti-TNF-R antibodies. 9-12 In particular pentameric IgM anti-TNF-R antibodies or cross-linked, monoclonal anti-TNF-R IgG antibodies are effective in eliciting biological activities in several cell lines. The trimeric TNF molecule contains three receptor-binding sites, located in the clefts between the subunits. 13 Indeed, the stoichiometry of binding in solution for the soluble, extracellular domains of TNF-R55 or TNF-R75 to the homotrimeric TNF molecule ranged between 2 and 3. 14-16 Lymphotoxin is closely related to TNF and binds to the same receptors. The X-ray structure of the soluble human (h) TNF-R55/human lymphotoxin complex confirmed the previous data and showed three receptor molecules bound to one lymphotoxin trimer. 17
FEBS Letters, 1996
In order to map the immunogenic epitope for the produced mainly by activated monocytes-macrophages, was monoclonal antibody E7H2 on the human turnout necrosis factor initially characterized as a protein inducing the haemorrhagic (hTNF-~) molecule, a number of chimeric proteins were necrosis of certain transplanted tumours in mice [7]. To date, developed by in-frame joining segments of the human genes TNF-c~ has been proved to be a key mediator of inflammation encoding TNF-c~ and lymphotoxin (TNF-[3) as well as by during infectious diseases and exerts strong system effects incoupling appropriate coding regions for human and mouse cluding the septic shock [8]. In an attempt to gain more in-TNF-c~. High level expression of these chimeric genes was sight into the functional topography of the TNF-~ molecule, achieved in Escherichia coli by placing the coding sequences we initiated an epitope analysis using monoclonal antibodies under control of either E. coli trp-promoter or a tandem of (Mabs) E7H2 [9] which are specific for human recombinant bacteriophage T7 constitutive promoters A2 and A3. As revealed TNF-~ [10]. Here, we report the construction of chimeric and by Western blot analysis with monoclonal antibody E7H2 mutant TNF molecules and their use for defining the epitope directed against human TNF-~, the region involved in the binding of this antibody includes sequence ValGluLeuArg in the recognized by Mabs E7H2. N-terminal part of the TNF-ot molecule. 2. Materials and methods
Journal of Biological Chemistry, 2012
Background: Human TNF-␣ is a cytokine involved in many disease-related cellular processes. Results: High affinity binding of therapeutic antibody (inhibitor) to native and molten globule-like TNF-␣ conformation is driven by specific noncovalent interactions. Conclusion: Binding-coupled conformational changes are crucial for antibody-TNF-␣ recognition. Significance: This work helps learn which forces drive unfolding of TNF-␣ and its recognition by monoclonal antibodies and how they affect TNF-␣ activity regulation.
Modifying TNF alpha for Therapeutic Use A Perspective on the TNF Receptor System
TNF alpha is an inflammatory mediator that is relevant to several autoimmune diseases. Macromolecular inhibitors of TNF alpha have proven therapeutically useful in some preliminary studies. We have developed small molecule TNF alpha antagonist based on the crystal structure of TNF receptor complex. The TNF alpha inhibitor is specific and mediates biological function similar to the inhibitory soluble TNF receptor. This review focuses on development of small molecule anti-TNF alpha mimetics by us and current status of other agents.
Unraveling the Binding Mechanism of Trivalent Tumor Necrosis Factor Ligands and Their Receptors
Molecular & Cellular Proteomics, 2011
Characterization of the binding of a tumor necrosis factor (TNF) ligand to its receptor(s) is pivotal to understand how these proteins initiate signal transduction pathways. Unfortunately, kinetic elucidation of these interactions is strongly hampered by the multivalent nature of the binding partners. The interaction between TNF-related apoptosis-inducing ligand and its death receptors was analyzed using in-depth applications of surface plasmon resonance technology. Variations in receptor density and sensor chip type allowed us to manipulate the stoichiometry of the formed complex, and the rate constants describing the binding of trimeric TNF-related apoptosisinducing ligand to only one receptor molecule were determined. Remarkably, the affinity of this trimer-monomer complex is in the picomolar range, and its dissociation very slow. Further analysis showed that the second and third receptor molecules bind with lower affinity to the preformed trimer-monomer complex. This together with results obtained with receptor activator of NF-B ligand and B cell-activating factor strongly suggests that the binding of TNF family ligands to their receptors is initiated via the formation of a trimer-monomer complex that is sufficiently stable to allow binding of two additional receptor molecules. These results suggest that avidity does not play a significant role and thus provide new insight in how TNF ligands form the biologically important complexes with their receptors. Molecular & Cellular Proteomics 10: 10.1074/mcp.M110.002808, 1-10, 2011.