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PDBsum entry 2b0d

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protein dna_rna metals Protein-protein interface(s) links
Hydrolase/DNA PDB id
2b0d
Jmol
Contents
Protein chains
236 a.a.
DNA/RNA
Metals
_CA ×2
Waters ×262
PDB id:
2b0d
Name: Hydrolase/DNA
Title: Ecorv restriction endonuclease/gaattc/ca2+
Structure: 5'-d( Ap Ap Ap Gp Ap Ap Tp Tp Cp Tp T)-3'. Chain: c, d. Engineered: yes. Type ii restriction enzyme ecorv. Chain: a, b. Synonym: endonuclease ecorv, r.Ecorv. Engineered: yes
Source: Synthetic: yes. Escherichia coli. Organism_taxid: 562. Gene: ecorvr. Expressed in: escherichia coli. Expression_system_taxid: 562.
Biol. unit: Tetramer (from PQS)
Resolution:
2.00Å     R-factor:   0.220     R-free:   0.302
Authors: D.A.Hiller,A.M.Rodriguez,J.J.Perona
Key ref:
D.A.Hiller et al. (2005). Non-cognate enzyme-DNA complex: structural and kinetic analysis of EcoRV endonuclease bound to the EcoRI recognition site GAATTC. J Mol Biol, 354, 121-136. PubMed id: 16236314 DOI: 10.1016/j.jmb.2005.09.046
Date:
13-Sep-05     Release date:   27-Sep-05    
PROCHECK
Go to PROCHECK summary
 Headers
 References

Protein chains
Pfam   ArchSchema ?
P04390  (T2E5_ECOLX) -  Type-2 restriction enzyme EcoRV
Seq:
Struc:
245 a.a.
236 a.a.
Key:    PfamA domain  Secondary structure  CATH domain

 Gene Ontology (GO) functional annotation 
  GO annot!
  Biological process     nucleic acid phosphodiester bond hydrolysis   3 terms 
  Biochemical function     hydrolase activity     6 terms  

 

 
DOI no: 10.1016/j.jmb.2005.09.046 J Mol Biol 354:121-136 (2005)
PubMed id: 16236314  
 
 
Non-cognate enzyme-DNA complex: structural and kinetic analysis of EcoRV endonuclease bound to the EcoRI recognition site GAATTC.
D.A.Hiller, A.M.Rodriguez, J.J.Perona.
 
  ABSTRACT  
 
The crystal structure of EcoRV endonuclease bound to non-cognate DNA at 2.0 angstroms resolution shows that very small structural adaptations are sufficient to ensure the extreme sequence specificity characteristic of restriction enzymes. EcoRV bends its specific GATATC site sharply by 50 degrees into the major groove at the center TA step, generating unusual base-base interactions along each individual DNA strand. In the symmetric non-cognate complex bound to GAATTC, the center step bend is relaxed to avoid steric hindrance caused by the different placement of the exocyclic thymine methyl groups. The decreased base-pair unstacking in turn leads to small conformational rearrangements in the sugar-phosphate backbone, sufficient to destabilize binding of crucial divalent metal ions in the active site. A second crystal structure of EcoRV bound to the base-analog GAAUTC site shows that the 50 degrees center-step bend of the DNA is restored. However, while divalent metals bind at high occupancy in this structure, one metal ion shifts away from binding at the scissile DNA phosphate to a position near the 3'-adjacent phosphate group. This may explain why the 10(4)-fold attenuated cleavage efficiency toward GAATTC is reconstituted by less than tenfold toward GAAUTC. Examination of DNA binding and bending by equilibrium and stopped-flow florescence quenching and fluorescence resonance energy transfer (FRET) methods demonstrates that the capacity of EcoRV to bend the GAATTC non-cognate site is severely limited, but that full bending of GAAUTC is achieved at only a threefold reduced rate compared with the cognate complex. Together, the structural and biochemical data demonstrate the existence of distinct mechanisms for ensuring specificity at the bending and catalytic steps, respectively. The limited conformational rearrangements observed in the EcoRV non-cognate complex provide a sharp contrast to the extensive structural changes found in a non-cognate BamHI-DNA crystal structure, thus demonstrating a diversity of mechanisms by which restriction enzymes are able to achieve specificity.
 
  Selected figure(s)  
 
Figure 5.
Figure 5. (a) Superposition of the recognition loops (residues 180-190 shown) of TA (green), AT (red) and AU (blue). DNA from TA is shown in grey. In AU, the loops from each monomer have moved apart approximately 0.6 Å. (b) van der Waals contacts made by Thr186 to the center step of TA. The C^g-methyl of Thr186 (blue) lies in a pocket formed by the C5-methyl of thymine (orange), the O4 of thymine and the N6 of adenine (red). (c) Center step contacts in AT, shown as in (b). Even when adenine and thymine are switched in AT, a similar set of hydrophobic contacts is made.
Figure 7.
Figure 7. Metal binding sites in TA (a), AT (b), and AU (c). Only one metal, bound to site II, is seen in the P1 lattice for TA. AT has two metal ions bound in one subunit, with high B-factors. AU also has two metal ions bound in one subunit. One metal is bound to site I, which is also seen in other modified complexes with poor activity.
 
  The above figures are reprinted by permission from Elsevier: J Mol Biol (2005, 354, 121-136) copyright 2005.  
  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.  
18838672 B.van den Broek, M.A.Lomholt, S.M.Kalisch, R.Metzler, and G.J.Wuite (2008).
How DNA coiling enhances target localization by proteins.
  Proc Natl Acad Sci U S A, 105, 15738-15742.  
17430971 M.Saravanan, K.Vasu, R.Kanakaraj, D.N.Rao, and V.Nagaraja (2007).
R.KpnI, an HNH superfamily REase, exhibits differential discrimination at non-canonical sequences in the presence of Ca2+ and Mg2+.
  Nucleic Acids Res, 35, 2777-2786.  
17437717 S.A.Townson, J.C.Samuelson, Y.Bao, S.Y.Xu, and A.K.Aggarwal (2007).
BstYI bound to noncognate DNA reveals a "hemispecific" complex: implications for DNA scanning.
  Structure, 15, 449-459.
PDB code: 2p0j
16845123 B.Youngblood, and N.O.Reich (2006).
Conformational transitions as determinants of specificity for the DNA methyltransferase EcoRI.
  J Biol Chem, 281, 26821-26831.  
16981705 D.A.Hiller, and J.J.Perona (2006).
Positively charged C-terminal subdomains of EcoRV endonuclease: contributions to DNA binding, bending, and cleavage.
  Biochemistry, 45, 11453-11463.
PDB code: 2ge5
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