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PDBsum entry 1az4

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protein Protein-protein interface(s) links
Endonuclease PDB id
1az4
Jmol
Contents
Protein chains
187 a.a.
215 a.a.
Waters ×42
PDB id:
1az4
Name: Endonuclease
Title: Ecorv endonuclease, unliganded, form b, t93a mutant
Structure: Ecorv endonuclease. Chain: a, b. Ec: 3.1.21.4
Source: Escherichia coli. Organism_taxid: 562
Resolution:
2.40Å     R-factor:   0.225     R-free:   0.337
Authors: J.Perona,A.Martin
Key ref:
J.J.Perona and A.M.Martin (1997). Conformational transitions and structural deformability of EcoRV endonuclease revealed by crystallographic analysis. J Mol Biol, 273, 207-225. PubMed id: 9367757 DOI: 10.1006/jmbi.1997.1315
Date:
24-Nov-97     Release date:   27-May-98    
PROCHECK
Go to PROCHECK summary
 Headers
 References

Protein chain
Pfam   ArchSchema ?
P04390  (T2E5_ECOLX) -  Type-2 restriction enzyme EcoRV
Seq:
Struc:
245 a.a.
187 a.a.*
Protein chain
Pfam   ArchSchema ?
P04390  (T2E5_ECOLX) -  Type-2 restriction enzyme EcoRV
Seq:
Struc:
245 a.a.
215 a.a.*
Key:    PfamA domain  Secondary structure  CATH domain
* PDB and UniProt seqs differ at 2 residue positions (black crosses)

 Enzyme reactions 
   Enzyme class: Chains A, B: 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     6 terms  

 

 
DOI no: 10.1006/jmbi.1997.1315 J Mol Biol 273:207-225 (1997)
PubMed id: 9367757  
 
 
Conformational transitions and structural deformability of EcoRV endonuclease revealed by crystallographic analysis.
J.J.Perona, A.M.Martin.
 
  ABSTRACT  
 
The structures of wild-type and mutant forms of the unliganded EcoRV endonuclease dimer have been determined at 2.4 A resolution in a new crystal lattice. Comparison of these structures with that of the free enzyme determined with different packing constraints shows that the conformations of the domain interfaces are not conserved between crystal forms. The unliganded enzyme and the enzyme-DNA complex delineate two distinct quaternary states separated by a 25 degrees intersubunit rotation, but considerable conformational heterogeneity, of the order of 10 degrees domain rotations, exists within each of these states. Comparison of the free enzyme structure between the two crystal forms further reveals that the C-terminal 28 amino acid residues are disordered and undergo an extensive local folding transition upon DNA binding. Introduction of the mutation T93A at the DNA-binding cleft causes large-scale effects on the protein conformation. Structural changes in the mutated unliganded enzyme propagate some 20 to 25 A to the dimerization interface and lead to a rearrangement of monomer subunits. Comparative analysis of these structures, a new structure of the enzyme cocrystallized with DNA and calcium ions, and previously determined cocrystal structures suggests important roles for a number of amino acid residues in facilitating the intersubunit motions and local folding transitions. In particular, the T93A structure reveals a pathway through the protein, by which DNA-binding may cause the domain movements required for proper alignment of catalytic groups. The key active-site residue Glu45 is located on a flexible helix inside this pathway, and this provides a direct means by which essential catalytic functions are coupled to the protein conformational change. It appears that indirect perturbation of the Glu45 conformation via an altered quaternary structure may be a contributing factor to the decreased catalytic efficiency of T93A, and this mechanism may also explain the diminished activities of other active site variants of EcoRV.
 
  Selected figure(s)  
 
Figure 7.
Figure 7. (a) Ribbon representation of the crystal struc- ture of the EcoRV-DNA complex showing the location of residue Thr93 in the DNA binding cleft, and its pos- ition with respect to helix B and the dimer interface region (DIM). (b) Stereo view of a superposition of WTUCB (yellow) and T93A (orange) using polypeptide backbone atoms within one of the DNA-binding domains, as defined by difference-distance calculations. A steric clash between Phe47 of T93A (orange) and Val20 of WTUCB (yellow) shows the origin of the domain reorientation in the mutant.
Figure 8.
Figure 8. (a) Stereo view of the active site of the ternary complex of EcoRV cocrystallized with cognate DNA and Ca 2+ . The position of the leaving 30 oxygen atom of the scissile bond is indicated. In this conformation, in-line attack of the hydroxide ion to form the expected trigonal bipyramid in the transition state must be from the face opposite the position of the carboxylate groups. The calcium ion (#283 CL) binds between Asp90, Asp74 and the scissile phos- phate groups. Helix B is at the left. (b) Detailed view of the ligation of the calcium ion in subunit I of the EcoRV- DNA-Ca 2+ complex, including distances (in Å ) as determined from the final refined coordinate set. The scissile phos- phate group is shown at the top. The ligation of Ca 2+ in subunit II is very similar, except that electron density for one apical water molecule is not apparent in electron density maps, and the two oxygen atoms of Asp90 are located in the equatorial plane at distances of 2.53 Å and 2.46 Å from the calcium ion.
 
  The above figures are reprinted by permission from Elsevier: J Mol Biol (1997, 273, 207-225) copyright 1997.  
  Figures were selected by an automated process.  

Literature references that cite this PDB file's key reference

  PubMed id Reference
19596810 S.R.Bellamy, Y.S.Kovacheva, I.H.Zulkipli, and S.E.Halford (2009).
Differences between Ca2+ and Mg2+ in DNA binding and release by the SfiI restriction endonuclease: implications for DNA looping.
  Nucleic Acids Res, 37, 5443-5453.  
17307879 B.M.Reinhard, S.Sheikholeslami, A.Mastroianni, A.P.Alivisatos, and J.Liphardt (2007).
Use of plasmon coupling to reveal the dynamics of DNA bending and cleavage by single EcoRV restriction enzymes.
  Proc Natl Acad Sci U S A, 104, 2667-2672.  
17223133 S.de los Rios, and J.J.Perona (2007).
Structure of the Escherichia coli leucine-responsive regulatory protein Lrp reveals a novel octameric assembly.
  J Mol Biol, 366, 1589-1602.
PDB code: 2gqq
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
16498623 K.J.Skowronek, J.Kosinski, and J.M.Bujnicki (2006).
Theoretical model of restriction endonuclease HpaI in complex with DNA, predicted by fold recognition and validated by site-directed mutagenesis.
  Proteins, 63, 1059-1068.  
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.  
14634013 A.J.Noël, W.Wende, and A.Pingoud (2004).
DNA recognition by the homing endonuclease PI-SceI involves a divalent metal ion cofactor-induced conformational change.
  J Biol Chem, 279, 6794-6804.  
15210696 B.B.Hopkins, and N.O.Reich (2004).
Simultaneous DNA binding, bending, and base flipping: evidence for a novel M.EcoRI methyltransferase-DNA complex.
  J Biol Chem, 279, 37049-37060.  
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.  
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.  
12142452 M.Fuxreiter, and I.Simon (2002).
Protein stability indicates divergent evolution of PD-(D/E)XK type II restriction endonucleases.
  Protein Sci, 11, 1978-1983.  
11842098 S.Grazulis, M.Deibert, R.Rimseliene, R.Skirgaila, G.Sasnauskas, A.Lagunavicius, V.Repin, C.Urbanke, R.Huber, and V.Siksnys (2002).
Crystal structure of the Bse634I restriction endonuclease: comparison of two enzymes recognizing the same DNA sequence.
  Nucleic Acids Res, 30, 876-885.
PDB code: 1knv
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.  
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
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.  
10103058 C.M.Dupureur, and L.M.Hallman (1999).
Effects of divalent metal ions on the activity and conformation of native and 3-fluorotyrosine-PvuII endonucleases.
  Eur J Biochem, 261, 261-268.  
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.  
9822618 C.Schulze, A.Jeltsch, I.Franke, C.Urbanke, and A.Pingoud (1998).
Crosslinking the EcoRV restriction endonuclease across the DNA-binding site reveals transient intermediates and conformational changes of the enzyme during DNA binding and catalytic turnover.
  EMBO J, 17, 6757-6766.  
  9628339 F.Stahl, W.Wende, A.Jeltsch, and A.Pingoud (1998).
The mechanism of DNA cleavage by the type II restriction enzyme EcoRV: Asp36 is not directly involved in DNA cleavage but serves to couple indirect readout to catalysis.
  Biol Chem, 379, 467-473.  
9548954 F.Stahl, W.Wende, C.Wenz, A.Jeltsch, and A.Pingoud (1998).
Intra- vs intersubunit communication in the homodimeric restriction enzyme EcoRV: Thr 37 and Lys 38 involved in indirect readout are only important for the catalytic activity of their own subunit.
  Biochemistry, 37, 5682-5688.  
9783752 H.Viadiu, and A.K.Aggarwal (1998).
The role of metals in catalysis by the restriction endonuclease BamHI.
  Nat Struct Biol, 5, 910-916.
PDB codes: 2bam 3bam
9705308 N.C.Horton, and J.J.Perona (1998).
Recognition of flanking DNA sequences by EcoRV endonuclease involves alternative patterns of water-mediated contacts.
  J Biol Chem, 273, 21721-21729.
PDB code: 1bgb
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
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.