2zjc Citations

Structure-function relationship of tumor necrosis factor (TNF) and its receptor interaction based on 3D structural analysis of a fully active TNFR1-selective TNF mutant.


Tumor necrosis factor (TNF) is an important cytokine that suppresses carcinogenesis and excludes infectious pathogens to maintain homeostasis. TNF activates its two receptors [TNF receptor (TNFR) 1 and TNFR2], but the contribution of each receptor to various host defense functions and immunologic surveillance is not yet clear. Here, we used phage display techniques to generate receptor-selective TNF mutants that activate only one TNFR. These TNF mutants will be useful in the functional analysis of TNFR. Six amino acids in the receptor binding interface (near TNF residues 30, 80, and 140) were randomly mutated by polymerase chain reaction. Two phage libraries comprising over 5 million TNF mutants were constructed. By selecting the mutants without affinity for TNFR1 or TNFR2, we successfully isolated 4 TNFR2-selective candidates and 16 TNFR1-selective candidates, respectively. The TNFR1-selective candidates were highly mutated near residue 30, whereas TNFR2-selective candidates were highly mutated near residue 140, although both had conserved sequences near residues 140 and 30, respectively. This finding suggested that the phage display technique was suitable for identifying important regions for the TNF interaction with TNFR1 and TNFR2. Purified clone R1-6, a TNFR1-selective candidate, remained fully bioactive and had full affinity for TNFR1 without activating TNFR2, indicating the usefulness of the R1-6 TNF mutant in analyzing TNFR1 receptor function. To further elucidate the receptor selectivity of R1-6, we examined the structure of R1-6 by X-ray crystallography. The results suggested that R31A and R32G mutations strongly influenced electrostatic interaction with TNFR2, and that L29K mutation contributed to the binding of R1-6 to TNFR1. This phage display technique can be used to efficiently construct functional mutants for analysis of the TNF structure-function relationship, which might facilitate in silico drug design based on receptor selectivity.

Reviews citing this publication (8)

  1. Tumor Necrosis Factor-Alpha and Pregnancy: Focus on Biologics. An Updated and Comprehensive Review. Alijotas-Reig J, Esteve-Valverde E, Ferrer-Oliveras R, Llurba E, Gris JM. Clin Rev Allergy Immunol 53 40-53 (2017)
  2. Development of novel drug delivery systems using phage display technology for clinical application of protein drugs. Nagano K, Tsutsumi Y. Proc Jpn Acad Ser B Phys Biol Sci 92 156-166 (2016)
  3. Regulation of TNF-α with a focus on rheumatoid arthritis. Moelants EA, Mortier A, Van Damme J, Proost P. Immunol Cell Biol 91 393-401 (2013)
  4. Cytokines and epilepsy. Li G, Bauer S, Nowak M, Norwood B, Tackenberg B, Rosenow F, Knake S, Oertel WH, Hamer HM. Seizure 20 249-256 (2011)
  5. Tumor necrosis factor-alpha as a potential therapeutic target in idiopathic inflammatory myopathies. Stübgen JP. J Neurol 258 961-970 (2011)
  6. [Creation of TNFR1-selective antagonist and its therapeutic effects]. Nomura T, Abe Y, Yoshioka Y, Nakagawa S, Tsunoda S, Tsutsumi Y. Yakugaku Zasshi 130 63-68 (2010)
  7. Engineering signal transduction pathways. Kiel C, Yus E, Serrano L. Cell 140 33-47 (2010)
  8. TNF receptor 2 pathway: drug target for autoimmune diseases. Faustman D, Davis M. Nat Rev Drug Discov 9 482-493 (2010)

Articles citing this publication (23)

  1. Elucidating glycosaminoglycan-protein-protein interactions using carbohydrate microarray and computational approaches. Rogers CJ, Clark PM, Tully SE, Abrol R, Garcia KC, Goddard WA, Hsieh-Wilson LC. Proc Natl Acad Sci U S A 108 9747-9752 (2011)
  2. Comparison of the inhibition mechanisms of adalimumab and infliximab in treating tumor necrosis factor α-associated diseases from a molecular view. Hu S, Liang S, Guo H, Zhang D, Li H, Wang X, Yang W, Qian W, Hou S, Wang H, Guo Y, Lou Z. J Biol Chem 288 27059-27067 (2013)
  3. Structural basis for treating tumor necrosis factor α (TNFα)-associated diseases with the therapeutic antibody infliximab. Liang S, Dai J, Hou S, Su L, Zhang D, Guo H, Hu S, Wang H, Rao Z, Guo Y, Lou Z. J Biol Chem 288 13799-13807 (2013)
  4. Enriching the human apoptosis pathway by predicting the structures of protein-protein complexes. Acuner Ozbabacan SE, Keskin O, Nussinov R, Gursoy A. J Struct Biol 179 338-346 (2012)
  5. Crystal structure of TNFalpha complexed with a poxvirus MHC-related TNF binding protein. Yang Z, West AP, Bjorkman PJ. Nat Struct Mol Biol 16 1189-1191 (2009)
  6. Citrullination of TNF-α by peptidylarginine deiminases reduces its capacity to stimulate the production of inflammatory chemokines. Moelants EA, Mortier A, Grauwen K, Ronsse I, Van Damme J, Proost P. Cytokine 61 161-167 (2013)
  7. Unraveling the binding mechanism of trivalent tumor necrosis factor ligands and their receptors. Reis CR, van Assen AH, Quax WJ, Cool RH. Mol Cell Proteomics 10 M110.002808 (2011)
  8. Correlating RANK ligand/RANK binding kinetics with osteoclast formation and function. Warren JT, Zou W, Decker CE, Rohatgi N, Nelson CA, Fremont DH, Teitelbaum SL. J Cell Biochem 116 2476-2483 (2015)
  9. Crystallization and preliminary X-ray analysis of the tumour necrosis factor alpha-tumour necrosis factor receptor type 2 complex. Mukai Y, Nakamura T, Yoshioka Y, Tsunoda S, Kamada H, Nakagawa S, Yamagata Y, Tsutsumi Y. Acta Crystallogr Sect F Struct Biol Cryst Commun 65 295-298 (2009)
  10. Fast binding kinetics and conserved 3D structure underlie the antagonistic activity of mutant TNF: useful information for designing artificial proteo-antagonists. Mukai Y, Nakamura T, Yoshioka Y, Shibata H, Abe Y, Nomura T, Taniai M, Ohta T, Nakagawa S, Tsunoda S, Kamada H, Yamagata Y, Tsutsumi Y. J Biochem 146 167-172 (2009)
  11. A novel recombinant slow-release TNF α-derived peptide effectively inhibits tumor growth and angiogensis. Ma Y, Zhao S, Shen S, Fang S, Ye Z, Shi Z, Hong A. Sci Rep 5 13595 (2015)
  12. Apoptosis through Death Receptors in Temporal Lobe Epilepsy-Associated Hippocampal Sclerosis. Teocchi MA, D'Souza-Li L. Mediators Inflamm 2016 8290562 (2016)
  13. Novel protein engineering strategy for creating highly receptor-selective mutant TNFs. Nomura T, Abe Y, Kamada H, Inoue M, Kawara T, Arita S, Furuya T, Yoshioka Y, Shibata H, Kayamuro H, Yamashita T, Nagano K, Yoshikawa T, Mukai Y, Nakagawa S, Taniai M, Ohta T, Tsunoda S, Tsutsumi Y. Biochem Biophys Res Commun 388 667-671 (2009)
  14. Generation of mouse macrophages expressing membrane-bound TNF variants with selectivity for TNFR1 or TNFR2. Shibata H, Abe Y, Yoshioka Y, Nomura T, Sato M, Kayamuro H, Kawara T, Arita S, Furuya T, Nagano K, Yoshikawa T, Kamada H, Tsunoda S, Tsutsumi Y. Cytokine 50 75-83 (2010)
  15. Novel mutants of human tumor necrosis factor with dominant-negative properties. Shingarova LN, Boldyreva EF, Yakimov SA, Guryanova SV, Dolgikh DA, Nedospasov SA, Kirpichnikov MP. Biochemistry (Mosc) 75 1458-1463 (2010)
  16. Rational design of TNFα binding proteins based on the de novo designed protein DS119. Zhu C, Zhang C, Zhang T, Zhang X, Shen Q, Tang B, Liang H, Lai L. Protein Sci 25 2066-2075 (2016)
  17. [Production and properties of human tumor necrosis factor peptide fragments] Shingarova LN, Petrovskaia LE, Nekrasov AN, Kriukova EA, Boldyreva EF, Iakimov SA, Gur'ianova SV, Dolgikh DA, Kirpichnikov MP. Bioorg Khim 36 327-336 (2010)
  18. Deep brain stimulation of the anterior nucleus of the thalamus reverses the gene expression of cytokines and their receptors as well as neuronal degeneration in epileptic rats. Chen YC, Zhu GY, Wang X, Shi L, Jiang Y, Zhang X, Zhang JG. Brain Res 1657 304-311 (2017)
  19. Structural modeling of tumor necrosis factor: A protein of immunological importance. Roy U. Biotechnol Appl Biochem 64 454-463 (2017)
  20. NF-κB signaling and cell-fate decision induced by a fast-dissociating tumor necrosis factor mutant. Zhang X, Yin N, Guo A, Zhang Q, Zhang Y, Xu Y, Liu H, Tang B, Lai L. Biochem Biophys Res Commun 489 287-292 (2017)
  21. Novel RANKL DE-loop mutants antagonize RANK-mediated osteoclastogenesis. Wang Y, van Assen AHG, Reis CR, Setroikromo R, van Merkerk R, Boersma YL, Cool RH, Quax WJ. FEBS J 284 2501-2512 (2017)
  22. Functionality of intrinsic disorder in tumor necrosis factor-α and its receptors. Uversky VN, El-Baky NA, El-Fakharany EM, Sabry A, Mattar EH, Uversky AV, Redwan EM. FEBS J 284 3589-3618 (2017)
  23. Creation of mouse TNFR2-selective agonistic TNF mutants using a phage display technique. Ando D, Inoue M, Kamada H, Taki S, Furuya T, Abe Y, Nagano K, Tsutsumi Y, Tsunoda SI. Biochem Biophys Rep 7 309-315 (2016)