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PDBsum entry 2zjc

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protein ligands Protein-protein interface(s) links
Cytokine PDB id
2zjc
Jmol
Contents
Protein chains
142 a.a. *
151 a.a. *
Ligands
GOL
Waters ×59
* Residue conservation analysis
PDB id:
2zjc
Name: Cytokine
Title: Tnfr1 selectve tnf mutant; r1-6
Structure: Tumor necrosis factor. Chain: a, b, c. Fragment: r1-6, unp residues 77-233. Synonym: tnf-alpha, tumor necrosis factor ligand superfamil 2, tnf-a, cachectin. Engineered: yes. Mutation: yes
Source: Homo sapiens. Human. Organism_taxid: 9606. Gene: tnf, tnfa, tnfsf2. Expressed in: escherichia coli. Expression_system_taxid: 562.
Resolution:
2.50Å     R-factor:   0.209     R-free:   0.272
Authors: Y.Mukai,Y.Yamagata,Y.Tsutsumi
Key ref:
Y.Mukai et al. (2009). 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. J Mol Biol, 385, 1221-1229. PubMed id: 19084540 DOI: 10.1016/j.jmb.2008.11.053
Date:
05-Mar-08     Release date:   20-Jan-09    
PROCHECK
Go to PROCHECK summary
 Headers
 References

Protein chains
Pfam   ArchSchema ?
P01375  (TNFA_HUMAN) -  Tumor necrosis factor
Seq:
Struc:
233 a.a.
142 a.a.*
Protein chain
Pfam   ArchSchema ?
P01375  (TNFA_HUMAN) -  Tumor necrosis factor
Seq:
Struc:
233 a.a.
151 a.a.*
Key:    PfamA domain  Secondary structure  CATH domain
* PDB and UniProt seqs differ at 22 residue positions (black crosses)

 Gene Ontology (GO) functional annotation 
  GO annot!
  Cellular component     membrane   1 term 
  Biological process     immune response   1 term 
  Biochemical function     tumor necrosis factor receptor binding     1 term  

 

 
DOI no: 10.1016/j.jmb.2008.11.053 J Mol Biol 385:1221-1229 (2009)
PubMed id: 19084540  
 
 
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.
Y.Mukai, H.Shibata, T.Nakamura, Y.Yoshioka, Y.Abe, T.Nomura, M.Taniai, T.Ohta, S.Ikemizu, S.Nakagawa, S.Tsunoda, H.Kamada, Y.Yamagata, Y.Tsutsumi.
 
  ABSTRACT  
 
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.
 
  Selected figure(s)  
 
Figure 1.
Fig. 1. Positions of randomized residues on the binding interface of the TNF–TNFR1 complex. Mutational residues of Library I (red spheres) and Library II (orange spheres). Green cartoon represents wtTNF. White area represents the surface of the TNFR1 monomer. This binding model structure of the TNF–TNFR1 complex was constructed based on the crystal structure of the LTα–TNFR1 complex (1TNR) and that of wtTNF (1TNF).
Figure 5.
Fig. 5. Model of TNF binding to TNFR1 and TNFR2. Receptor binding interfaces of (a) wtTNF–TNFR1 (green–red); (b) R1-6–TNFR1 (white–red); (c) wtTNF–TNFR2 (green–blue); and (d) R1-6–TNFR2 (white–blue). The TNF–TNFR1 model complex was constructed from 1TNF (wtTNF) and 1TNR (LTα–TNFR1 complex). The predicted TNFR2 structure was constructed by side-chain mutation using the O program. In this simulation, the side chains of each structure were rotated to fit the predicted interaction. Stable structures of these rotamers were constructed using the O program. Steric hindrance might have occurred between His69 of TNFR1 and Arg32 of wtTNF in (a) (black arrowhead). Potential interactions are indicated by orange arrows. A cluster of anionic charged residues (Asp54, Glu57, and Glu70) is highlighted by a broken red line.
 
  The above figures are reprinted by permission from Elsevier: J Mol Biol (2009, 385, 1221-1229) copyright 2009.  
  Figures were selected by an automated process.  

Literature references that cite this PDB file's key reference

  PubMed id Reference
20085704 C.Kiel, E.Yus, and L.Serrano (2010).
Engineering signal transduction pathways.
  Cell, 140, 33-47.  
20489699 D.Faustman, and M.Davis (2010).
TNF receptor 2 pathway: drug target for autoimmune diseases.
  Nat Rev Drug Discov, 9, 482-493.  
21314616 L.N.Shingarova, E.F.Boldyreva, S.A.Yakimov, S.V.Guryanova, D.A.Dolgikh, S.A.Nedospasov, and M.P.Kirpichnikov (2010).
Novel mutants of human tumor necrosis factor with dominant-negative properties.
  Biochemistry (Mosc), 75, 1458-1463.  
20046067 T.Nomura, Y.Abe, Y.Yoshioka, S.Nakagawa, S.Tsunoda, and Y.Tsutsumi (2010).
[Creation of TNFR1-selective antagonist and its therapeutic effects]
  Yakugaku Zasshi, 130, 63-68.  
19386778 Y.Mukai, T.Nakamura, Y.Yoshioka, H.Shibata, Y.Abe, T.Nomura, M.Taniai, T.Ohta, S.Nakagawa, S.Tsunoda, H.Kamada, Y.Yamagata, and Y.Tsutsumi (2009).
Fast binding kinetics and conserved 3D structure underlie the antagonistic activity of mutant TNF: useful information for designing artificial proteo-antagonists.
  J Biochem, 146, 167-172.
PDB code: 2zpx
  19255488 Y.Mukai, T.Nakamura, Y.Yoshioka, S.Tsunoda, H.Kamada, S.Nakagawa, Y.Yamagata, and Y.Tsutsumi (2009).
Crystallization and preliminary X-ray analysis of the tumour necrosis factor alpha-tumour necrosis factor receptor type 2 complex.
  Acta Crystallogr Sect F Struct Biol Cryst Commun, 65, 295-298.  
19838188 Z.Yang, A.P.West, and P.J.Bjorkman (2009).
Crystal structure of TNFalpha complexed with a poxvirus MHC-related TNF binding protein.
  Nat Struct Mol Biol, 16, 1189-1191.  
The most recent references are shown first. Citation data come partly from CiteXplore and partly from an automated harvesting procedure. Note that this is likely to be only a partial list as not all journals are covered by either method. However, we are continually building up the citation data so more and more references will be included with time. Where a reference describes a PDB structure, the PDB code is shown on the right.