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PDBsum entry 2oxv

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protein dna_rna links
Hydrolase/DNA PDB id
2oxv
Jmol
Contents
Protein chain
261 a.a.
DNA/RNA
Waters ×136
PDB id:
2oxv
Name: Hydrolase/DNA
Title: Structure of the a138t promiscuous mutant of the ecori restriction endonuclease bound to its cognate recognition site.
Structure: Type ii restriction enzyme ecori. Chain: a. Synonym: endonuclease ecori, r.Ecori. Engineered: yes. Mutation: yes. DNA (5'- d( Tp Cp Gp Cp Gp Ap Ap Tp Tp Cp Gp Cp G)-3'). Chain: c. Engineered: yes
Source: Escherichia coli. Organism_taxid: 562. Gene: ecorir. Expressed in: escherichia coli bl21(de3). Expression_system_taxid: 469008. Synthetic: yes. Other_details: abi3900 DNA synthesizer
Resolution:
1.95Å     R-factor:   0.202     R-free:   0.244
Authors: P.J.Sapienza,J.M.Rosenberg,L.Jen-Jacobson
Key ref:
P.J.Sapienza et al. (2007). Structural and thermodynamic basis for enhanced DNA binding by a promiscuous mutant EcoRI endonuclease. Structure, 15, 1368-1382. PubMed id: 17997963 DOI: 10.1016/j.str.2007.09.014
Date:
21-Feb-07     Release date:   23-Oct-07    
PROCHECK
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 Headers
 References

Protein chain
Pfam   ArchSchema ?
P00642  (T2E1_ECOLX) -  Type-2 restriction enzyme EcoRI
Seq:
Struc:
277 a.a.
261 a.a.*
Key:    PfamA domain  Secondary structure  CATH domain
* PDB and UniProt seqs differ at 1 residue position (black cross)

 Enzyme reactions 
   Enzyme class: E.C.3.1.21.4  - Type Ii site-specific deoxyribonuclease.
[IntEnz]   [ExPASy]   [KEGG]   [BRENDA]
      Reaction: Endonucleolytic cleavage of DNA to give specific double-stranded fragments with terminal 5'-phosphates.
      Cofactor: Mg(2+)
 Gene Ontology (GO) functional annotation 
  GO annot!
  Biological process     nucleic acid phosphodiester bond hydrolysis   3 terms 
  Biochemical function     hydrolase activity     7 terms  

 

 
DOI no: 10.1016/j.str.2007.09.014 Structure 15:1368-1382 (2007)
PubMed id: 17997963  
 
 
Structural and thermodynamic basis for enhanced DNA binding by a promiscuous mutant EcoRI endonuclease.
P.J.Sapienza, J.M.Rosenberg, L.Jen-Jacobson.
 
  ABSTRACT  
 
Promiscuous mutant EcoRI endonucleases bind to the canonical site GAATTC more tightly than does the wild-type endonuclease, yet cleave variant (EcoRI(*)) sites more rapidly than does wild-type. The crystal structure of the A138T promiscuous mutant homodimer in complex with a GAATTC site is nearly identical to that of the wild-type complex, except that the Thr138 side chains make packing interactions with bases in the 5'-flanking regions outside the recognition hexanucleotide while excluding two bound water molecules seen in the wild-type complex. Molecular dynamics simulations confirm exclusion of these waters. The structure and simulations suggest possible reasons why binding of the A138T protein to the GAATTC site has DeltaS degrees more favorable and DeltaH degrees less favorable than for wild-type endonuclease binding. The interactions of Thr138 with flanking bases may permit A138T, unlike wild-type enzyme, to form complexes with EcoRI(*) sites that structurally resemble the specific wild-type complex with GAATTC.
 
  Selected figure(s)  
 
Figure 2.
Figure 2. Effects of the A138T Mutation on Water-Mediated Contacts to Bases Flanking the GAATTC Recognition Site
2F[o] − F[c] simulated annealing omit maps covering the wild-type (A) and A138T (B) structures in the vicinity of G[−5]C[−4]. Maps are contoured at 1.5 σ in both panels, with mesh-covering protein in red and DNA in blue. Water molecules are shown as blue spheres and hydrogen bonds are depicted as black lines in all panels. (C) and (D) show space-filled renderings of the same views given in (A) and (B). Note the novel water-mediated interactions between Thr138 and G[−5], the packing interactions of the Thr138 γ-methyl group with C[−4]-N4 and G[−5]-N7, and the absence of water41 in the mutant model.
Figure 3.
Figure 3. Water Network at the 5′ Side of the Recognition Site
(A) Wild-type model. Coupling between water1, which contacts G[−3] in the recognition site, and water41, which contacts the flanking base C[−4]. Note that Arg200-N epsilon interacts with water1 and Arg200-NH1 with water41. Water1 is involved in similar contacts in the A138T structure. Arg200-NH1 is hydrogen bonded to Asn199 and Gly196 carbonyls (not shown).
(B) A138T model. The position and contacts of water1, water74, and water149 are conserved despite the absence of water41.
(C) Water oxygen to DNA functional group distances calculated over the course of a 5 ns MD simulation of the wild-type-d(TCGCGAATTCGCG) complex at 300K. The plots refer to sites occupied by water molecules in positions analogous to water41 (black) and water1 (red) in the X-ray model. The plot shows that (1) the water41 binding site occupied in the 100K model is also occupied in the 300K complex, and (2) the residence time of water41 is similar to that of a solvent molecule that is buried at the GAATTC recognition site.
(D) Radial distribution functions (g[x]) for water oxygens relative to the 4-amino group of C[−4]. Data are from wild-type (black) and A138T (red) simulations. The A138T plot shows no peak within hydrogen-bonding distance, so this binding site is not occupied during the 300K simulation of the A138T complex. Plots (B) and (C) are for one half-site only, but results are typical of both half-sites.
 
  The above figures are reprinted by permission from Cell Press: Structure (2007, 15, 1368-1382) copyright 2007.  
  Figures were selected by an automated process.  

Literature references that cite this PDB file's key reference

  PubMed id Reference
  18541926 M.T.Langhans, and M.J.Palladino (2009).
Cleavage of mispaired heteroduplex DNA substrates by numerous restriction enzymes.
  Curr Issues Mol Biol, 11, 1.  
  19021169 K.M.Stone, J.E.Townsend, J.Sarver, P.J.Sapienza, S.Saxena, and L.Jen-Jacobson (2008).
Electron spin resonance shows common structural features for different classes of EcoRI-DNA complexes.
  Angew Chem Int Ed Engl, 47, 10192-10194.  
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.