PDBsum entry 1fxy

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Hydrolase/hydrolase inhibitor PDB id
Protein chain
228 a.a. *
Waters ×82
* Residue conservation analysis
PDB id:
Name: Hydrolase/hydrolase inhibitor
Title: Coagulation factor xa-trypsin chimera inhibited with d-phe-p chloromethylketone
Structure: Coagulation factor xa-trypsin chimera. Chain: a. Engineered: yes. Mutation: yes
Source: Homo sapiens. Human. Organism_taxid: 9606. Expressed in: escherichia coli. Expression_system_taxid: 562.
Biol. unit: Tetramer (from PQS)
2.15Å     R-factor:   0.180     R-free:   0.245
Authors: K.P.Hopfner,E.Kopetzki,G.-B.Kresse,R.Huber,W.Bode,R.A.Engh
Key ref:
K.P.Hopfner et al. (1998). New enzyme lineages by subdomain shuffling. Proc Natl Acad Sci U S A, 95, 9813-9818. PubMed id: 9707558 DOI: 10.1073/pnas.95.17.9813
22-Apr-98     Release date:   17-Jun-98    
Go to PROCHECK summary

Protein chain
Pfam   ArchSchema ?
P07477  (TRY1_HUMAN) -  Trypsin-1
247 a.a.
228 a.a.*
Key:    PfamA domain  Secondary structure  CATH domain
* PDB and UniProt seqs differ at 69 residue positions (black crosses)

 Enzyme reactions 
   Enzyme class: E.C.  - Trypsin.
[IntEnz]   [ExPASy]   [KEGG]   [BRENDA]
      Reaction: Preferential cleavage: Arg-|-Xaa, Lys-|-Xaa.
 Gene Ontology (GO) functional annotation 
  GO annot!
  Biological process     proteolysis   1 term 
  Biochemical function     catalytic activity     2 terms  


DOI no: 10.1073/pnas.95.17.9813 Proc Natl Acad Sci U S A 95:9813-9818 (1998)
PubMed id: 9707558  
New enzyme lineages by subdomain shuffling.
K.P.Hopfner, E.Kopetzki, G.B.Kresse, W.Bode, R.Huber, R.A.Engh.
Protein functions have evolved in part via domain recombination events. Such events, for example, recombine structurally independent functional domains and shuffle targeting, regulatory, and/or catalytic functions. Domain recombination, however, can generate new functions, as implied by the observation of catalytic sites at interfaces of distinct folding domains. If useful to an evolving organism, such initially rudimentary functions would likely acquire greater efficiency and diversity, whereas the initially distinct folding domains would likely develop into single functional domains. This represents the probable evolution of the S1 serine protease family, whose two homologous beta-barrel subdomains assemble to form the binding sites and the catalytic machinery. Among S1 family members, the contact interface and catalytic residues are highly conserved whereas surrounding surfaces are highly variable. This observation suggests a new strategy to engineer viable proteins with novel properties, by swapping folding subdomains chosen from among protein family members. Such hybrid proteins would retain properties conserved throughout the family, including folding stability as single domain proteins, while providing new surfaces amenable to directed evolution or engineering of specific new properties. We show here that recombining the N-terminal subdomain from coagulation factor X with the C-terminal subdomain from trypsin creates a potent enzyme (fXYa) with novel properties, in particular a broad substrate specificity. As shown by the 2.15-A crystal structure, plasticity at the hydrophobic subdomain interface maintains activity, while surface loops are displaced compared with the parent subdomains. fXYa thus represents a new serine proteinase lineage with hybrid fX, trypsin, and novel properties.
  Selected figure(s)  
Figure 2.
Fig. 2. Crystal structure of the fXa/trypsin hybrid. Ribbon plot of the crystal structure of fXYa. The N-terminal subdomain is shown in red, and the C-terminal subdomain is shown in yellow. Both subdomains adopt a -barrel fold and assemble asymmetrically to generate the fold typical of the chymotrypsin family. Disulfide bridges are depicted in green (the N-terminal bridge discussed in the text is located behind the C-terminal barrel). The D-Phe-Pro-Arg inhibitor is shown with magenta sticks. It is bound to the active site, which is formed at the subdomain interface. The catalytic triad residue side chains are displayed explicitly as sticks.
Figure 3.
Fig. 3. Comparison of the active site of fXYa to fXa and trypsin. Final model of fXYa showing the active site (thick sticks using the color code of Fig. 2) with representative 1.0 contoured 2Fc-Fo electron density for the catalytic tetrad residues His52, Asp102, Ser195, and Ser214 as well as for the PPACK inhibitor. The N-terminal subdomain of fXYa (red) is superimposed with the N-terminal subdomain of fXa (blue); the C-terminal subdomain (yellow) is superimposed with the C-terminal subdomain of trypsin (green). The structure shows a well conserved catalytic triad and specificity pocket. Some side chain adjustments in substrate binding sites (S1-S3) presumably originate from interaction with PPACK.
  Figures were selected by an automated process.  

Literature references that cite this PDB file's key reference

  PubMed id Reference
19165722 Y.Chen, S.Li, T.Chen, H.Hua, and Z.Lin (2009).
Random dissection to select for protein split sites and its application in protein fragment complementation.
  Protein Sci, 18, 399-409.  
18391948 N.Varadarajan, S.Rodriguez, B.Y.Hwang, G.Georgiou, and B.L.Iverson (2008).
Highly active and selective endopeptidases with programmed substrate specificities.
  Nat Chem Biol, 4, 290-294.  
17180359 M.A.Moreno-Risueno, M.Martínez, J.Vicente-Carbajosa, and P.Carbonero (2007).
The family of DOF transcription factors: from green unicellular algae to vascular plants.
  Mol Genet Genomics, 277, 379-390.  
10975452 M.Olsen, B.Iverson, and G.Georgiou (2000).
High-throughput screening of enzyme libraries.
  Curr Opin Biotechnol, 11, 331-337.  
10975450 S.Lutz, and S.J.Benkovic (2000).
Homology-independent protein engineering.
  Curr Opin Biotechnol, 11, 319-324.  
11087392 Y.Liu, L.A.Witucki, K.Shah, A.C.Bishop, and K.M.Shokat (2000).
Src-Abl tyrosine kinase chimeras: replacement of the adenine binding pocket of c-Abl with v-Src to swap nucleotide and inhibitor specificities.
  Biochemistry, 39, 14400-14408.  
10102985 H.Czapinska, and J.Otlewski (1999).
Structural and energetic determinants of the S1-site specificity in serine proteases.
  Eur J Biochem, 260, 571-595.  
10467148 K.P.Hopfner, A.Lang, A.Karcher, K.Sichler, E.Kopetzki, H.Brandstetter, R.Huber, W.Bode, and R.A.Engh (1999).
Coagulation factor IXa: the relaxed conformation of Tyr99 blocks substrate binding.
  Structure, 7, 989-996.
PDB code: 1rfn
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.