PDBsum entry 2b0d

Hydrolase/DNA

PDB id: 2b0d

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

Protein chains

P04390  (T2E5_ECOLX) -  Type II restriction enzyme EcoRV from Escherichia coli

Seq: 245 a.a.

Struc: 236 a.a.

DNA/RNA chains

A-A-A-G-A-A-T-T-C-T-T 11 bases

A-A-A-G-A-A-T-T-C-T-T 11 bases

Enzyme reactions

• Enzyme class: E.C.3.1.21.4 - type Ii site-specific deoxyribonuclease.
• Reaction: Endonucleolytic cleavage of DNA to give specific double-stranded fragments with terminal 5'-phosphates.
• Cofactor: Mg(2+)

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.