PDBsum entry 1brc

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protein Protein-protein interface(s) links
Complex(proteinase/inhibitor) PDB id
Protein chains
223 a.a. *
56 a.a. *
Waters ×141
* Residue conservation analysis
PDB id:
Name: Complex(proteinase/inhibitor)
Title: Relocating a negative charge in the binding pocket of trypsin
Structure: Trypsin. Chain: e. Engineered: yes. Amyloid beta-protein precursor inhibitor domain (appi). Chain: i. Engineered: yes
Source: Rattus norvegicus. Norway rat. Organism_taxid: 10116. Expressed in: unidentified. Expression_system_taxid: 32644.
Biol. unit: Tetramer (from PQS)
2.50Å     R-factor:   0.168    
Authors: J.J.Perona,R.J.Fletterick
Key ref: J.J.Perona et al. (1993). Relocating a negative charge in the binding pocket of trypsin. J Mol Biol, 230, 934-949. PubMed id: 8478942
17-Dec-92     Release date:   31-May-94    
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Protein chain
Pfam   ArchSchema ?
P00763  (TRY2_RAT) -  Anionic trypsin-2
246 a.a.
223 a.a.*
Protein chain
Pfam   ArchSchema ?
P05067  (A4_HUMAN) -  Amyloid beta A4 protein
770 a.a.
56 a.a.
Key:    PfamA domain  PfamB domain  Secondary structure  CATH domain
* PDB and UniProt seqs differ at 2 residue positions (black crosses)

 Gene Ontology (GO) functional annotation 
  GO annot!
  Cellular component     cellular_component   3 terms 
  Biological process     digestion   4 terms 
  Biochemical function     catalytic activity     8 terms  


J Mol Biol 230:934-949 (1993)
PubMed id: 8478942  
Relocating a negative charge in the binding pocket of trypsin.
J.J.Perona, C.A.Tsu, M.E.McGrath, C.S.Craik, R.J.Fletterick.
The functional and structural consequences of altering the position of the negatively charged aspartate residue at the base of the specificity pocket of trypsin have been examined by site-directed mutagenesis, kinetic characterization and crystallographic analysis. Anionic rat trypsin D189G/G226D exhibits a high level of catalytic activity on activated amide substrates, but its relative preference for lysine versus arginine as the P1 site residue is shifted by 30 to 40-fold in favor of lysine. The crystal structure of this variant has been determined in complexes with BPTI (bovine pancreatic trypsin inhibitor), APPI (amyloid beta-protein precursor inhibitor domain) and benzamidine inhibitors, at resolutions of 2.1 A, 2.5 A and 2.2 A, respectively. Asp226 bridges the base of the specificity pocket with its negative charge partially buried by interactions made with Ser190 and Tyr228. An equal reduction in the affinity of the variant enzyme for Arg and Lys substrates is attributable to a decreased electrostatic interaction of each ligand with the relocated aspartate residue. Comparison of structural and functional parameters with those of wild-type trypsin suggests that direct hydrogen-bonding electrostatic contacts in the S1 site do not significantly improve the free energy of substrate binding relative to indirect water-mediated interactions. The conformation adopted by Asp226, as well as by other adjacent side-chain and backbone groups, depends upon the ligand bound in the primary specificity pocket. This structural flexibility may be of critical importance to the retention of catalytic activity by the variant enzyme.

Literature references that cite this PDB file's key reference

  PubMed id Reference
16924626 P.F.Gratzer, J.P.Santerre, and J.M.Lee (2007).
The effect of chemical modification of amino acid side-chains on collagen degradation by enzymes.
  J Biomed Mater Res B Appl Biomater, 81, 1.  
16672242 T.T.Baird, W.D.Wright, and C.S.Craik (2006).
Conversion of trypsin to a functional threonine protease.
  Protein Sci, 15, 1229-1238.  
16041079 A.Schmidt, and V.S.Lamzin (2005).
Extraction of functional motion in trypsin crystal structures.
  Acta Crystallogr D Biol Crystallogr, 61, 1132-1139.
PDB codes: 1xvm 1xvo
16021597 R.Villar, M.J.Gil, J.I.García, and V.Martínez-Merino (2005).
Are AM1 ligand-protein binding enthalpies good enough for use in the rational design of new drugs?
  J Comput Chem, 26, 1347-1358.  
16038610 T.S.Zamolodchikova, E.V.Smirnova, A.N.Andrianov, I.V.Kashparov, O.D.Kotsareva, E.A.Sokolova, K.B.Ignatov, and A.D.Pemberton (2005).
Cloning and molecular modeling of duodenase with respect to evolution of substrate specificity within mammalian serine proteases that have lost a conserved active-site disulfide bond.
  Biochemistry (Mosc), 70, 672-684.  
14741623 P.F.Gratzer, J.P.Santerre, and J.M.Lee (2004).
Modulation of collagen proteolysis by chemical modification of amino acid side-chains in acellularized arteries.
  Biomaterials, 25, 2081-2094.  
10637221 H.Jing, Y.Xu, M.Carson, D.Moore, K.J.Macon, J.E.Volanakis, and S.V.Narayana (2000).
New structural motifs on the chymotrypsin fold and their potential roles in complement factor B.
  EMBO J, 19, 164-173.
PDB code: 1dle
10692303 M.R.Gunner, M.A.Saleh, E.Cross, A.ud-Doula, and M.Wise (2000).
Backbone dipoles generate positive potentials in all proteins: origins and implications of the effect.
  Biophys J, 78, 1126-1144.  
10617628 Y.Xu, A.Circolo, H.Jing, Y.Wang, S.V.Narayana, and J.E.Volanakis (2000).
Mutational analysis of the primary substrate specificity pocket of complement factor B. Asp(226) is a major structural determinant for p(1)-Arg binding.
  J Biol Chem, 275, 378-385.  
10382669 A.Caputo, J.C.Parrish, M.N.James, J.C.Powers, and R.C.Bleackley (1999).
Electrostatic reversal of serine proteinase substrate specificity.
  Proteins, 35, 415-424.  
  10210204 A.Pasternak, D.Ringe, and L.Hedstrom (1999).
Comparison of anionic and cationic trypsinogens: the anionic activation domain is more flexible in solution and differs in its mode of BPTI binding in the crystal structure.
  Protein Sci, 8, 253-258.
PDB codes: 3tgi 3tgj
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.  
9811833 G.O.Reznik, S.Vajda, T.Sano, and C.R.Cantor (1998).
A streptavidin mutant with altered ligand-binding specificity.
  Proc Natl Acad Sci U S A, 95, 13525-13530.  
9675278 J.Polanowska, I.Krokoszynska, H.Czapinska, W.Watorek, M.Dadlez, and J.Otlewski (1998).
Specificity of human cathepsin G.
  Biochim Biophys Acta, 1386, 189-198.  
9811827 N.C.Horton, K.J.Newberry, and J.J.Perona (1998).
Metal ion-mediated substrate-assisted catalysis in type II restriction endonucleases.
  Proc Natl Acad Sci U S A, 95, 13489-13494.
PDB code: 1bss
9374470 J.J.Perona, and C.S.Craik (1997).
Evolutionary divergence of substrate specificity within the chymotrypsin-like serine protease fold.
  J Biol Chem, 272, 29987-29990.  
18629959 T.Makriyannis, and Y.D.Clonis (1997).
Design and study of peptide-ligand affinity chromatography adsorbents: Application to the case of trypsin purification from bovine pancreas.
  Biotechnol Bioeng, 53, 49-57.  
9370374 T.S.Zamolodchikova, E.A.Sokolova, S.L.Alexandrov, I.I.Mikhaleva, I.A.Prudchenko, I.A.Morozov, N.V.Kononenko, O.A.Mirgorodskaya, U.Da, N.I.Larionova, V.F.Pozdnev, D.Ghosh, W.L.Duax, and T.I.Vorotyntseva (1997).
Subcellular localization, substrate specificity and crystallization of duodenase, a potential activator of enteropeptidase.
  Eur J Biochem, 249, 612-621.  
  8931142 D.H.Shin, H.K.Song, I.S.Seong, C.S.Lee, C.H.Chung, and S.W.Suh (1996).
Crystal structure analyses of uncomplexed ecotin in two crystal forms: implications for its function and stability.
  Protein Sci, 5, 2236-2247.
PDB codes: 1ecy 1ecz
  7795518 J.J.Perona, and C.S.Craik (1995).
Structural basis of substrate specificity in the serine proteases.
  Protein Sci, 4, 337-360.
PDB code: 1amh
  7757004 M.E.McGrath, S.A.Gillmor, and R.J.Fletterick (1995).
Ecotin: lessons on survival in a protease-filled world.
  Protein Sci, 4, 141-148.  
7664048 A.Caputo, M.N.James, J.C.Powers, D.Hudig, and R.C.Bleackley (1994).
Conversion of the substrate specificity of mouse proteinase granzyme B.
  Nat Struct Biol, 1, 364-367.  
  8156987 M.E.McGrath, T.Erpel, C.Bystroff, and R.J.Fletterick (1994).
Macromolecular chelation as an improved mechanism of protease inhibition: structure of the ecotin-trypsin complex.
  EMBO J, 13, 1502-1507.  
7527150 Q.Zheng, and D.J.Kyle (1994).
Multiple copy sampling: rigid versus flexible protein.
  Proteins, 19, 324-329.  
8020486 V.Tõugu, T.Tiivel, P.Talts, V.Siksnis, S.Poyarkova, T.Kesvatera, and A.Aaviksaar (1994).
Electrostatic effects in trypsin reactions. Influence of salts.
  Eur J Biochem, 222, 475-481.  
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 codes are shown on the right.