PDBsum entry 1bua

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protein dna_rna Protein-protein interface(s) links
Hydrolase/DNA PDB id
Protein chains
231 a.a.
229 a.a.
Waters ×236
PDB id:
Name: Hydrolase/DNA
Title: Structural and energetic origins of indirect readout in site-specific DNA cleavage by a restriction endonuclease
Structure: DNA (5'-d( Ap Ap Ap Gp Ap Cp Ip Tp Cp Tp T)-3'). Chain: c, d. Engineered: yes. Endonuclease ecorv. Chain: a, b. Engineered: yes
Source: Synthetic: yes. Escherichia coli. Organism_taxid: 562. Expressed in: escherichia coli. Expression_system_taxid: 562
Biol. unit: Tetramer (from PQS)
2.15Å     R-factor:   0.200     R-free:   0.288
Authors: J.J.Perona,A.M.Martin
Key ref:
A.M.Martin et al. (1999). Structural and energetic origins of indirect readout in site-specific DNA cleavage by a restriction endonuclease. Nat Struct Biol, 6, 269-277. PubMed id: 10074946 DOI: 10.1038/6707
03-Sep-98     Release date:   09-Sep-98    
Go to PROCHECK summary

Protein chain
Pfam   ArchSchema ?
P04390  (T2E5_ECOLX) -  Type-2 restriction enzyme EcoRV
245 a.a.
231 a.a.
Protein chain
Pfam   ArchSchema ?
P04390  (T2E5_ECOLX) -  Type-2 restriction enzyme EcoRV
245 a.a.
229 a.a.
Key:    PfamA domain  Secondary structure  CATH domain

 Enzyme reactions 
   Enzyme class: Chains A, B: E.C.  - 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: Magnesium
 Gene Ontology (GO) functional annotation 
  GO annot!
  Biological process     nucleic acid phosphodiester bond hydrolysis   3 terms 
  Biochemical function     hydrolase activity     6 terms  


DOI no: 10.1038/6707 Nat Struct Biol 6:269-277 (1999)
PubMed id: 10074946  
Structural and energetic origins of indirect readout in site-specific DNA cleavage by a restriction endonuclease.
A.M.Martin, M.D.Sam, N.O.Reich, J.J.Perona.
Specific recognition by EcoRV endonuclease of its cognate, sharply bent GATATC site at the center TA step occurs solely via hydrophobic interaction with thymine methyl groups. Mechanistic kinetic analyses of base analog-substituted DNAs at this position reveal that direct readout provides 5 kcal mol(-1) toward specificity, with an additional 6-10 kcal mol(-1) arising from indirect readout. Crystal structures of several base analog complexes show that the major-groove hydrophobic contacts are crucial to forming required divalent metal-binding sites, and that indirect readout operates in part through the sequence-dependent free-energy cost of unstacking the center base-pair step of the DNA.
  Selected figure(s)  
Figure 1.
Figure 1. a, Ribbon representation of the EcoRV dimer showing the dimerization domain at bottom (orange), the DNA-binding/catalytic domains in yellow and the four flexible linkers I−IV in each subunit^13. The major-groove binding R-loops (red, residues 182−188 of each subunit) present all of the primary determinants for direct readout of base-pair functional groups in the GATATC target. The scissile phosphorus atoms are shown by the pink spheres. The Gln-rich Q-loops that bind in the minor groove are shown in blue. b, View of the bent DNA conformation as seen in the complex of EcoRV with specific DNA^13. The center TA step and the R-loops are drawn in purple and blue, respectively. Van der Waals contacts of the Thr 186 side-chain methyl groups (red) with functional groups in the major groove are shown. c, Hydrogen-bonding (dotted lines) and van der Waals/electrostatic contacts (hatched lines) at the center TA step in the wild-type EcoRV−DNA complex. Distances in Å between nonhydrogen atoms are from the ternary complex structure with Ca^2+ (ref. 15). The contacts between Thr 186 and the thymine O4/adenine N6 groups may contribute binding energy but are considered to be nonspecific. The Watson−Crick hydrogen bonds are designated WC. The distance between the Thr 186 and Thr 186' methyl groups from the two separate subunits is 4.2 Å.
Figure 4.
Figure 4. a, Superposition of the structures of EcoRV bound to the wild-type site in the presence of Ca^2+ (green), and bound to CI (red). The subunit II active site is shown, and the superposition is carried out over all backbone atoms in the core portion of this subunit^15. The position of the calcium ion (Ca) and its bound water molecules in the active site of the cognate structure are shown. b, 'Omit' (2F[o] - F[c]) electron density map in the active site of subunit I of the EcoRV−CI complex, contoured at 1.0 . Side chains of Asp 90, Asp 74, Glu 45 and Lys 92, the center CI step of the DNA, and all solvent molecules in the subunit I active site were removed before positional refinement in X-PLOR. Positions of side chains and water molecules (blue spheres) in the final model are shown. B-factors for the water molecules shown range from 30 to 35 Å^2. This and other maps through the course of refinement show the lack of a well-defined Ca^2+ ion in the active site. c, Simulated annealing 'omit' electron density map in the active site of subunit I of the EcoRV−MI−Ca^2+ complex. Side chains of Asp 90, Asp 74, Glu 45 and Lys 92, the center MI step of the DNA, and all solvent molecules in the subunit I active site were removed, and the resulting model subjected to a simulated annealing refinement protocol in X-PLOR. Electron density maps calculated with coefficients (2F[o] - F[c]) (blue) and (F[o] - F[c]) (red) are shown superimposed on the final model. Phases for this map were derived from the model with these atoms deleted. The map is computed in the resolution range from 2.0 Å to 20 Å. The density is contoured at 1.0 for the (2F[o] - F[c]) map and 6.0 for the (F[o] - F[c]) map. The purple sphere represents a Ca^2+ ion and blue spheres represent water molecules. Both map figures were produced using SETOR^46.
  The above figures are reprinted by permission from Macmillan Publishers Ltd: Nat Struct Biol (1999, 6, 269-277) copyright 1999.  
  Figures were selected by an automated process.  

Literature references that cite this PDB file's key reference Google scholar

  PubMed id Reference
20861000 M.Firczuk, M.Wojciechowski, H.Czapinska, and M.Bochtler (2011).
DNA intercalation without flipping in the specific ThaI-DNA complex.
  Nucleic Acids Res, 39, 744-754.
PDB code: 3ndh
19389725 J.Ashworth, and D.Baker (2009).
Assessment of the optimization of affinity and specificity at protein-DNA interfaces.
  Nucleic Acids Res, 37, e73.  
  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.  
19497854 R.A.Estabrook, T.T.Nguyen, N.Fera, and N.O.Reich (2009).
Coupling sequence-specific recognition to DNA modification.
  J Biol Chem, 284, 22690-22696.  
19081059 E.J.Little, A.C.Babic, and N.C.Horton (2008).
Early interrogation and recognition of DNA sequence by indirect readout.
  Structure, 16, 1828-1837.
PDB code: 3ebc
18653536 S.Lindemose, P.E.Nielsen, and N.E.Møllegaard (2008).
Dissecting direct and indirect readout of cAMP receptor protein DNA binding using an inosine and 2,6-diaminopurine in vitro selection system.
  Nucleic Acids Res, 36, 4797-4807.  
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
16675462 H.K.Joshi, C.Etzkorn, L.Chatwell, J.Bitinaite, and N.C.Horton (2006).
Alteration of sequence specificity of the type II restriction endonuclease HincII through an indirect readout mechanism.
  J Biol Chem, 281, 23852-23869.
PDB codes: 2gie 2gig 2gih 2gii 2gij
15886396 B.van den Broek, M.C.Noom, and G.J.Wuite (2005).
DNA-tension dependence of restriction enzyme activity reveals mechanochemical properties of the reaction pathway.
  Nucleic Acids Res, 33, 2676-2684.  
15375161 S.Chandrashekaran, M.Saravanan, D.R.Radha, and V.Nagaraja (2004).
Ca(2+)-mediated site-specific DNA cleavage and suppression of promiscuous activity of KpnI restriction endonuclease.
  J Biol Chem, 279, 49736-49740.  
15489861 S.Hauenstein, C.M.Zhang, Y.M.Hou, and J.J.Perona (2004).
Shape-selective RNA recognition by cysteinyl-tRNA synthetase.
  Nat Struct Mol Biol, 11, 1134-1141.
PDB code: 1u0b
14661948 D.A.Hiller, J.M.Fogg, A.M.Martin, J.M.Beechem, N.O.Reich, and J.J.Perona (2003).
Simultaneous DNA binding and bending by EcoRV endonuclease observed by real-time fluorescence.
  Biochemistry, 42, 14375-14385.  
11891293 H.Asahara, and O.C.Uhlenbeck (2002).
The tRNA specificity of Thermus thermophilus EF-Tu.
  Proc Natl Acad Sci U S A, 99, 3499-3504.  
11742344 N.C.Horton, L.F.Dorner, and J.J.Perona (2002).
Sequence selectivity and degeneracy of a restriction endonuclease mediated by DNA intercalation.
  Nat Struct Biol, 9, 42-47.
PDB code: 1kc6
11861910 Z.Morávek, S.Neidle, and B.Schneider (2002).
Protein and drug interactions in the minor groove of DNA.
  Nucleic Acids Res, 30, 1182-1191.  
11557805 A.Pingoud, and A.Jeltsch (2001).
Structure and function of type II restriction endonucleases.
  Nucleic Acids Res, 29, 3705-3727.  
11588263 F.J.LaRiviere, A.D.Wolfson, and O.C.Uhlenbeck (2001).
Uniform binding of aminoacyl-tRNAs to elongation factor Tu by thermodynamic compensation.
  Science, 294, 165-168.  
11327870 S.L.Reid, D.Parry, H.H.Liu, and B.A.Connolly (2001).
Binding and recognition of GATATC target sequences by the EcoRV restriction endonuclease: a study using fluorescent oligonucleotides and fluorescence polarization.
  Biochemistry, 40, 2484-2494.  
10801972 N.C.Horton, and J.J.Perona (2000).
Crystallographic snapshots along a protein-induced DNA-bending pathway.
  Proc Natl Acad Sci U S A, 97, 5729-5734.
PDB codes: 1eoo 1eop
10856254 Q.Huai, J.D.Colandene, Y.Chen, F.Luo, Y.Zhao, M.D.Topal, and H.Ke (2000).
Crystal structure of NaeI-an evolutionary bridge between DNA endonuclease and topoisomerase.
  EMBO J, 19, 3110-3118.
PDB code: 1ev7
10387089 A.M.Martin, N.C.Horton, S.Lusetti, N.O.Reich, and J.J.Perona (1999).
Divalent metal dependence of site-specific DNA binding by EcoRV endonuclease.
  Biochemistry, 38, 8430-8439.  
10350476 M.D.Sam, and J.J.Perona (1999).
Catalytic roles of divalent metal ions in phosphoryl transfer by EcoRV endonuclease.
  Biochemistry, 38, 6576-6586.  
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.