PDBsum entry 1bsu

Hydrolase/DNA

PDB id: 1bsu

Contents

Protein chains

236 a.a.

DNA/RNA

Metals

_CA ×2

Waters ×218

PDB id:

1bsu

Name:

Hydrolase/DNA

Title:

Structural and energetic origins of indirect readout in site-specific DNA cleavage by a restriction endonuclease

Structure:

DNA (5'-d(p Ap Ap Gp Ap (5Cm)p Ip Tp Cp Tp T)-3'). Chain: c. Engineered: yes. DNA (5'-d( Ap Ap Ap Gp Ap (5Cm)p Ip Tp Cp Tp T)-3'). Chain: d. Engineered: yes. Endonuclease ecorv (3.1.21.4). 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)

Resolution:

2.00Å R-factor: 0.183 R-free: 0.258

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

Date:

30-Aug-98 Release date: 09-Sep-98

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-G-A-5CM-I-T-C-T-T 10 bases

A-A-A-G-A-5CM-I-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: 10.1038/6707

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.

ABSTRACT

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.