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PDBsum entry 1gns

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Hydrolase PDB id
1gns
Jmol
Contents
Protein chain
263 a.a. *
Ligands
ACN
Waters ×155
* Residue conservation analysis
PDB id:
1gns
Name: Hydrolase
Title: Subtilisin bpn'
Structure: Subtilisin bpn'. Chain: a. Fragment: residues 111-181,191-382. Engineered: yes. Mutation: yes
Source: Bacillus amyloliquefaciens. Organism_taxid: 1390. Expressed in: escherichia coli. Expression_system_taxid: 469008.
Resolution:
1.80Å     R-factor:   0.165    
Authors: O.Almog,D.T.Gallagher,J.E.Ladner,S.Strausberg, P.Alexanderp.Bryan,G.L.Gilliland
Key ref:
O.Almog et al. (2002). Structural basis of thermostability. Analysis of stabilizing mutations in subtilisin BPN'. J Biol Chem, 277, 27553-27558. PubMed id: 12011071 DOI: 10.1074/jbc.M111777200
Date:
06-Oct-01     Release date:   27-Jun-02    
PROCHECK
Go to PROCHECK summary
 Headers
 References

Protein chain
Pfam   ArchSchema ?
P00782  (SUBT_BACAM) -  Subtilisin BPN'
Seq:
Struc:
382 a.a.
263 a.a.*
Key:    PfamA domain  Secondary structure  CATH domain
* PDB and UniProt seqs differ at 9 residue positions (black crosses)

 Enzyme reactions 
   Enzyme class: E.C.3.4.21.62  - Subtilisin.
[IntEnz]   [ExPASy]   [KEGG]   [BRENDA]
      Reaction: Hydrolysis of proteins with broad specificity for peptide bonds, and a preference for a large uncharged residue in P1. Hydrolyzes peptide amides.
 Gene Ontology (GO) functional annotation 
  GO annot!
  Biological process     proteolysis   1 term 
  Biochemical function     serine-type endopeptidase activity     1 term  

 

 
DOI no: 10.1074/jbc.M111777200 J Biol Chem 277:27553-27558 (2002)
PubMed id: 12011071  
 
 
Structural basis of thermostability. Analysis of stabilizing mutations in subtilisin BPN'.
O.Almog, D.T.Gallagher, J.E.Ladner, S.Strausberg, P.Alexander, P.Bryan, G.L.Gilliland.
 
  ABSTRACT  
 
The crystal structures of two thermally stabilized subtilisin BPN' variants, S63 and S88, are reported here at 1.8 and 1.9 A resolution, respectively. The micromolar affinity calcium binding site (site A) has been deleted (Delta75-83) in these variants, enabling the activity and thermostability measurements in chelating conditions. Each of the variants includes mutations known previously to increase the thermostability of calcium-independent subtilisin in addition to new stabilizing mutations. S63 has eight amino acid replacements: D41A, M50F, A73L, Q206W, Y217K, N218S, S221C, and Q271E. S63 has 75-fold greater stability than wild type subtilisin in chelating conditions (10 mm EDTA). The other variant, S88, has ten site-specific changes: Q2K, S3C, P5S, K43N, M50F, A73L, Q206C, Y217K, N218S, and Q271E. The two new cysteines form a disulfide bond, and S88 has 1000 times greater stability than wild type subtilisin in chelating conditions. Comparisons of the two new crystal structures (S63 in space group P2(1) with A cell constants 41.2, 78.1, 36.7, and beta = 114.6 degrees and S88 in space group P2(1)2(1)2(1) with cell constants 54.2, 60.4, and 82.7) with previous structures of subtilisin BPN' reveal that the principal changes are in the N-terminal region. The structural bases of the stabilization effects of the new mutations Q2K, S3C, P5S, D41A, Q206C, and Q206W are generally apparent. The effects are attributed to the new disulfide cross-link and to improved hydrophobic packing, new hydrogen bonds, and other rearrangements in the N-terminal region.
 
  Selected figure(s)  
 
Figure 1.
Fig. 1. Stereo superposition of 1SUP (gray), 1SUC (black), and S63 (white) crystal structures in the region of residue 41. The large gray sphere is the calcium ion that is coordinated by Asp-41 in the wild type 1SUP. In the absence of the calcium loop, the side chain reorients (black). The structure is then stabilized by truncating the side chain (white).
Figure 2.
Fig. 2. Stereo superposition of 1SUP (gray), 1SUC (black), and S63 (white) crystal structures showing the Q206W mutation. The Q206 side chain adopts different conformations in its two structures. Tyr-6 is also visible in its two conformations.
 
  The above figures are reprinted by permission from the ASBMB: J Biol Chem (2002, 277, 27553-27558) copyright 2002.  
  Figures were selected by an automated process.  

Literature references that cite this PDB file's key reference

  PubMed id Reference
19148779 F.Moradian, K.Khajeh, H.Naderi-Manesh, and M.Sadeghizadeh (2009).
Isolation, purification and characterization of a surfactants-, laundry detergents- and organic solvents-resistant alkaline protease from Bacillus sp. HR-08.
  Appl Biochem Biotechnol, 159, 33-45.  
18655058 O.Almog, A.González, N.Godin, M.de Leeuw, M.J.Mekel, D.Klein, S.Braun, G.Shoham, and R.L.Walter (2009).
The crystal structures of the psychrophilic subtilisin S41 and the mesophilic subtilisin Sph reveal the same calcium-loaded state.
  Proteins, 74, 489-496.
PDB codes: 2gko 2ixt 3d43
18951408 P.K.Mankoo, S.Sukumar, and R.Karchin (2009).
PIK3CA somatic mutations in breast cancer: Mechanistic insights from Langevin dynamics simulations.
  Proteins, 75, 499-508.  
19761257 T.Gallagher, B.Ruan, M.London, M.A.Bryan, and P.N.Bryan (2009).
Structure of a switchable subtilisin complexed with a substrate and with the activator azide.
  Biochemistry, 48, 10389-10394.  
17937401 O.Almog, A.Kogan, M.Leeuw, G.Y.Gdalevsky, R.Cohen-Luria, and A.H.Parola (2008).
Structural insights into cold inactivation of tryptophanase and cold adaptation of subtilisin S41.
  Biopolymers, 89, 354-359.  
17921497 C.Deutsch, and B.Krishnamoorthy (2007).
Four-body scoring function for mutagenesis.
  Bioinformatics, 23, 3009-3015.  
17386103 J.Liao, M.K.Warmuth, S.Govindarajan, J.E.Ness, R.P.Wang, C.Gustafsson, and J.Minshull (2007).
Engineering proteinase K using machine learning and synthetic genes.
  BMC Biotechnol, 7, 16.  
17446890 M.T.Reetz, and J.D.Carballeira (2007).
Iterative saturation mutagenesis (ISM) for rapid directed evolution of functional enzymes.
  Nat Protoc, 2, 891-903.  
15752367 P.Dürrschmidt, J.Mansfeld, and R.Ulbrich-Hofmann (2005).
An engineered disulfide bridge mimics the effect of calcium to protect neutral protease against local unfolding.
  FEBS J, 272, 1523-1534.  
15857780 V.G.Eijsink, S.Gåseidnes, T.V.Borchert, and B.van den Burg (2005).
Directed evolution of enzyme stability.
  Biomol Eng, 22, 21-30.  
15215524 R.Schultz-Heienbrok, T.Maier, and N.Sträter (2004).
Trapping a 96 degrees domain rotation in two distinct conformations by engineered disulfide bridges.
  Protein Sci, 13, 1811-1822.
PDB codes: 1oi8 1oid 1oie
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.