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PDBsum entry 2b0e

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Top Page protein dna_rna metals Protein-protein interface(s) links
Hydrolase/DNA PDB id
2b0e
Jmol
Contents
Protein chains
233 a.a.
DNA/RNA
Metals
_CA ×2
Waters ×253

References listed in PDB file
Key reference
Title Non-Cognate enzyme-Dna complex: structural and kinetic analysis of ecorv endonuclease bound to the ecori recognition site gaattc.
Authors D.A.Hiller, A.M.Rodriguez, J.J.Perona.
Ref. J Mol Biol, 2005, 354, 121-136. [DOI no: 10.1016/j.jmb.2005.09.046]
PubMed id 16236314
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.
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.
Secondary reference #1
Title Structural and energetic origins of indirect readout in site-Specific DNA cleavage by a restriction endonuclease.
Authors A.M.Martin, M.D.Sam, N.O.Reich, J.J.Perona.
Ref. Nat Struct Biol, 1999, 6, 269-277. [DOI no: 10.1038/6707]
PubMed id 10074946
Full text Abstract
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 reproduced from the cited reference with permission from Macmillan Publishers Ltd
Secondary reference #2
Title Simultaneous DNA binding and bending by ecorv endonuclease observed by real-Time fluorescence.
Authors D.A.Hiller, J.M.Fogg, A.M.Martin, J.M.Beechem, N.O.Reich, J.J.Perona.
Ref. Biochemistry, 2003, 42, 14375-14385. [DOI no: 10.1021/bi035520w]
PubMed id 14661948
Full text Abstract
Secondary reference #3
Title Dna cleavage by ecorv endonuclease: two metal ions in three metal ion binding sites.
Authors N.C.Horton, J.J.Perona.
Ref. Biochemistry, 2004, 43, 6841-6857. [DOI no: 10.1021/bi0499056]
PubMed id 15170321
Full text Abstract
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