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PDBsum entry 5tsw

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protein Protein-protein interface(s) links
Lymphokine PDB id
5tsw
Jmol
Contents
Protein chains
(+ 0 more) 149 a.a. *
Waters ×1068
* Residue conservation analysis
PDB id:
5tsw
Name: Lymphokine
Title: High resolution crystal structure of a human tnf-alpha mutant
Structure: Protein (tumor necrosis factor-alpha). Chain: a, b, c, d, e, f. Engineered: yes. Mutation: yes
Source: Homo sapiens. Human. Organism_taxid: 9606. Expressed in: escherichia coli bl21(de3). Expression_system_taxid: 469008.
Biol. unit: Hexamer (from PQS)
Resolution:
2.50Å     R-factor:   0.207     R-free:   0.299
Authors: S.-S.Cha,J.-S.Kim,H.-S.Cho,B.-H.Oh
Key ref:
S.S.Cha et al. (1998). High resolution crystal structure of a human tumor necrosis factor-alpha mutant with low systemic toxicity. J Biol Chem, 273, 2153-2160. PubMed id: 9442056 DOI: 10.1074/jbc.273.4.2153
Date:
22-Apr-99     Release date:   07-May-99    
Supersedes: 4tsw
PROCHECK
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 Headers
 References

Protein chains
Pfam   ArchSchema ?
P01375  (TNFA_HUMAN) -  Tumor necrosis factor
Seq:
Struc:
233 a.a.
149 a.a.*
Key:    PfamA domain  Secondary structure  CATH domain
* PDB and UniProt seqs differ at 3 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: 10.1074/jbc.273.4.2153 J Biol Chem 273:2153-2160 (1998)
PubMed id: 9442056  
 
 
High resolution crystal structure of a human tumor necrosis factor-alpha mutant with low systemic toxicity.
S.S.Cha, J.S.Kim, H.S.Cho, N.K.Shin, W.Jeong, H.C.Shin, Y.J.Kim, J.H.Hahn, B.H.Oh.
 
  ABSTRACT  
 
A human tumor necrosis factor-alpha (TNF-alpha) mutant (M3S) with low systemic toxicity in vivo was designed, and its structures in two different crystal packings were determined crystallographically at 1.8 and 2.15-A resolution, respectively, to explain altered biological activities of the mutant. M3S contains four changes: a hydrophilic substitution of L29S, two hydrophobic substitutions of S52I and Y56F, and a deletion of the N-terminal seven amino acids that is disordered in the structure of wild-type TNF-alpha. Compared with wild-type TNF-alpha, it exhibits 11- and 71-fold lower binding affinities for the human TNF-R55 and TNF-R75 receptors, respectively, and in vitro cytotoxic effect and in vivo systemic toxicity of M3S are 20 and 10 times lower, respectively. However, in a transplanted solid tumor mouse model, M3S suppresses tumor growth more efficiently than wild-type TNF-alpha. M3S is highly resistant to proteolysis by trypsin, and it exhibits increased thermal stability and a prolonged half-life in vivo. The L29S mutation causes substantial restructuring of the loop containing residues 29-36 into a rigid segment as a consequence of induced formation of intra- and intersubunit interactions, explaining the altered receptor binding affinity and thermal stability. A mass spectrometric analysis identified major proteolytic cleavage sites located on this loop, and thus the increased resistance of M3S to the proteolysis is consistent with the increased rigidity of the loop. The S52I and Y56F mutations do not induce a noticeable conformational change. The side chain of Phe56 projects into a hydrophobic cavity, while Ile52 is exposed to the bulk solvent. Ile52 should be involved in hydrophobic interactions with the receptors, since a mutant containing the same mutations as in M3S except for the L29S mutation exhibits an increased receptor binding affinity. The low systemic toxicity of M3S appears to be the effect of the reduced and selective binding affinities for the TNF receptors, and the superior tumor-suppression of M3S appears to be the effect of its weak but longer antitumoral activity in vivo compared with wild-type TNF-alpha. It is also expected that the 1.8-A resolution structure will serve as an accurate model for explaining the structure-function relationship of wild-type TNF-alpha and many TNF-alpha mutants reported previously and for the design of new TNF-alpha mutants.
 
  Selected figure(s)  
 
Figure 3.
Fig. 3. A hydrogen-bonded network induced by the I29S mutation. White dotted lines indicate hydrogen bonds. Water molecules are in magenta. Oxygen and nitrogen atoms are in red and yellow, respectively. Each subunit is represented by green and pink, respectively. Only the side chains involved in the interactions are shown for clarity.
Figure 8.
Fig. 8. Stereo diagram of the interactions of the loop containing Arg44 and of the loop containing Arg31 and Arg32. Arginine residues are in magenta, and hydrophobic side chains are in green. White dotted lines indicate hydrogen bonds. Oxygen and nitrogen atoms are in red and yellow, respectively. Only the^ side chains involved in the interactions are shown for clarity.
 
  The above figures are reprinted by permission from the ASBMB: J Biol Chem (1998, 273, 2153-2160) copyright 1998.  
  Figures were selected by an automated process.  

Literature references that cite this PDB file's key reference

  PubMed id Reference
  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.  
16052236 J.Gerspach, D.Müller, S.Münkel, O.Selchow, J.Nemeth, M.Noack, H.Petrul, A.Menrad, H.Wajant, and K.Pfizenmaier (2006).
Restoration of membrane TNF-like activity by cell surface targeting and matrix metalloproteinase-mediated processing of a TNF prodrug.
  Cell Death Differ, 13, 273-284.  
16636812 J.Gerspach, J.Németh, S.Münkel, H.Wajant, and K.Pfizenmaier (2006).
Target-selective activation of a TNF prodrug by urokinase-type plasminogen activator (uPA) mediated proteolytic processing at the cell surface.
  Cancer Immunol Immunother, 55, 1590-1600.  
14696119 Y.Liu, L.H.Cheung, J.W.Marks, and M.G.Rosenblum (2004).
Recombinant single-chain antibody fusion construct targeting human melanoma cells and containing tumor necrosis factor.
  Int J Cancer, 108, 549-557.  
11929488 E.Garbuzenko, A.Nagler, D.Pickholtz, P.Gillery, R.Reich, F.X.Maquart, and F.Levi-Schaffer (2002).
Human mast cells stimulate fibroblast proliferation, collagen synthesis and lattice contraction: a direct role for mast cells in skin fibrosis.
  Clin Exp Allergy, 32, 237-246.  
10862680 D.Y.Kim, J.Lee, V.Saraswat, and Y.H.Park (2000).
Glucagon-induced self-association of recombinant proteins in Escherichia coli and affinity purification using a fragment of glucagon receptor.
  Biotechnol Bioeng, 69, 418-428.  
10891884 H.T.Idriss, and J.H.Naismith (2000).
TNF alpha and the TNF receptor superfamily: structure-function relationship(s).
  Microsc Res Tech, 50, 184-195.  
10089307 K.J.Baeyens, H.L.De Bondt, A.Raeymaekers, W.Fiers, and C.J.De Ranter (1999).
The structure of mouse tumour-necrosis factor at 1.4 A resolution: towards modulation of its selectivity and trimerization.
  Acta Crystallogr D Biol Crystallogr, 55, 772-778.
PDB code: 2tnf
10216319 S.S.Cha, H.C.Shin, K.Y.Choi, and B.H.Oh (1999).
Expression, purification and crystallization of recombinant human TRAIL.
  Acta Crystallogr D Biol Crystallogr, 55, 1101-1104.  
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.