PDBsum entry 2vs8

DNA binding protein

PDB id: 2vs8

Contents

Protein chains

183 a.a. *

DNA/RNA

Ligands

ACT ×3

Metals

_MN ×6

Waters ×392

* Residue conservation analysis

PDB id:

2vs8

Name:

DNA binding protein

Title:

The crystal structure of i-dmoi in complex with DNA and mn

Structure:

Homing endonuclease i-dmoi. Chain: a, f, k. Fragment: residues 2-188. Synonym: i-dmoi. Engineered: yes. 5'-d( Gp Cp Cp Tp Tp Gp Cp Cp Gp Gp Gp Tp Ap A)-3'. Chain: b, g, l. Engineered: yes. 5'-d( Gp Tp Tp Cp Cp Gp Gp Cp Dgp Dcp Dgp)-3'.

Source:

Desulfurococcus mobilis. Organism_taxid: 2274. Expressed in: escherichia coli. Expression_system_taxid: 562. Synthetic: yes. Organism_taxid: 2274

Resolution:

2.10Å R-factor: 0.203 R-free: 0.248

Authors:

M.J.Marcaida,J.Prieto,P.Redondo,A.D.Nadra,A.Alibes,L.Serrano, S.Grizot,P.Duchateau,F.Paques,F.J.Blanco,G.Montoya

Key ref:

M.J.Marcaida et al. (2008). Crystal structure of I-DmoI in complex with its target DNA provides new insights into meganuclease engineering. Proc Natl Acad Sci U S A, 105, 16888-16893. PubMed id: 18974222 DOI: 10.1073/pnas.0804795105

Date:

21-Apr-08 Release date: 11-Nov-08

Protein chains

P21505  (DMO1_DESMO) -  Homing endonuclease I-DmoI from Desulfurococcus mucosus

Seq: 194 a.a.

Struc: 183 a.a.

DNA/RNA chains

G-C-C-T-T-G-C-C-G-G-G-T-A-A 14 bases

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

C-G-C-G-C-C-G-G-A-A-C-T-T-A-C 15 bases

C-C-G-G-C-A-A-G-G-C 10 bases

G-C-C-T-T-G-C-C-G-G-G-T-A-A 14 bases

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

C-G-C-G-C-C-G-G-A-A-C-T-T-A-C 15 bases

C-C-G-G-C-A-A-G-G-C 10 bases

G-C-C-T-T-G-C-C-G-G-G-T-A-A 14 bases

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

C-G-C-G-C-C-G-G-A-A-C-T-T-A-C 15 bases

C-C-G-G-C-A-A-G-G-C 10 bases

Enzyme reactions

• Enzyme class: E.C.3.1.-.-

DOI no: 10.1073/pnas.0804795105

Proc Natl Acad Sci U S A 105:16888-16893 (2008)

PubMed id: 18974222

Crystal structure of I-DmoI in complex with its target DNA provides new insights into meganuclease engineering.

M.J.Marcaida, J.Prieto, P.Redondo, A.D.Nadra, A.Alibés, L.Serrano, S.Grizot, P.Duchateau, F.Pâques, F.J.Blanco, G.Montoya.

ABSTRACT

Homing endonucleases, also known as meganucleases, are sequence-specific enzymes with large DNA recognition sites. These enzymes can be used to induce efficient homologous gene targeting in cells and plants, opening perspectives for genome engineering with applications in a wide series of fields, ranging from biotechnology to gene therapy. Here, we report the crystal structures at 2.0 and 2.1 A resolution of the I-DmoI meganuclease in complex with its substrate DNA before and after cleavage, providing snapshots of the catalytic process. Our study suggests that I-DmoI requires only 2 cations instead of 3 for DNA cleavage. The structure sheds light onto the basis of DNA binding, indicating key residues responsible for nonpalindromic target DNA recognition. In silico and in vivo analysis of the I-DmoI DNA cleavage specificity suggests that despite the relatively few protein-base contacts, I-DmoI is highly specific when compared with other meganucleases. Our data open the door toward the generation of custom endonucleases for targeted genome engineering using the monomeric I-DmoI scaffold.

Selected figure(s)

Figure 2.
Detailed view of the I-DmoI active site. (A and B) Anomalous difference maps illustrate the presence of only 1 atom of calcium in the DNA bound structure (A) and 2 manganese ions in the structure of the I-DmoI product complex (B). The protein is shown in yellow and the DNA in green. (C) Schematic diagram of the hypothetical enzymatic mechanism proposed for I-DmoI. Hydrolysis of the phosphodiester bonds would follow a 2-metal ion mechanism. The first metal ion (site1, colored in yellow in 1) is bound in 1 active site and the water nucleophile (colored in red) is positioned in the central site and can attack the coding strand (in 2). The regeneration of the central water together with a second metal ion in the second site (site2, colored in cyan in 3) would enable the second attack. The D21, G20 and E117, A116 are contributed by the LAGLIDADG motifs of the enzyme.

Figure 4.
Comparison of H-DreI and I-DmoI structures. (A) Cα trace superposition of I-DmoI and H-DreI in complex with their DNA. (B) Detailed view of the DNA moieties. (C and D) Detailed views of the loop1a and loop2a protein-DNA interactions. (E) Comparison of the helical and propeller twist values of I-DmoI and H-DreI DNAs.

Figures were selected by an automated process.