PDBsum entry 2ex5

Hydrolase/DNA

PDB id: 2ex5

Contents

Protein chains

207 a.a. *

DNA/RNA

Metals

_CA ×3

Waters ×263

* Residue conservation analysis

PDB id:

2ex5

Name:

Hydrolase/DNA

Title:

Group i intron-encoded homing endonuclease i-ceui complexed with DNA

Structure:

I-ceui DNA target site. Chain: x. Engineered: yes. I-ceui DNA target site, complementary strand. Chain: y. Engineered: yes. DNA endonuclease i-ceui. Chain: a, b. Synonym: 23s rrna intron 1 protein.

Source:

Synthetic: yes. Synthetic construct. Organism_taxid: 32630. Other_details: synthesized nucleic acid. Chlamydomonas eugametos. Organism_taxid: 3053. Gene: i-ceui. Expressed in: escherichia coli bl21(de3). Expression_system_taxid: 469008.

Biol. unit:

Tetramer (from PQS)

Resolution:

2.20Å R-factor: 0.219 R-free: 0.231

Authors:

P.C.Spiegel,B.L.Stoddard

Key ref:

P.C.Spiegel et al. (2006). The structure of I-CeuI homing endonuclease: Evolving asymmetric DNA recognition from a symmetric protein scaffold. Structure, 14, 869-880. PubMed id: 16698548 DOI: 10.1016/j.str.2006.03.009

Date:

07-Nov-05 Release date: 23-May-06

Protein chains

P32761  (DNE1_CHLMO) -  DNA endonuclease I-CeuI from Chlamydomonas moewusii

Seq: 218 a.a.

Struc: 207 a.a.*

* PDB and UniProt seqs differ at 1 residue position (black cross)

DNA/RNA chains

C-G-A-T-A-A-C-G-G-T-C-C-T-A-A-G-G-T-A-G-C-G-A-A-G-C 26 bases

G-C-T-T-C-G-C-T-A-C-C-T-T-A-G-G-A-C-C-G-T-T-A-T-C-G 26 bases

Enzyme reactions

• Enzyme class: E.C.3.1.-.-

DOI no: 10.1016/j.str.2006.03.009

Structure 14:869-880 (2006)

PubMed id: 16698548

The structure of I-CeuI homing endonuclease: Evolving asymmetric DNA recognition from a symmetric protein scaffold.

P.C.Spiegel, B.Chevalier, D.Sussman, M.Turmel, C.Lemieux, B.L.Stoddard.

ABSTRACT

Homing endonucleases are highly specific catalysts of DNA strand breaks, leading to the transfer of mobile intervening sequences containing the endonuclease ORF. We have determined the structure and DNA recognition behavior of I-CeuI, a homodimeric LAGLIDADG endonuclease from Chlamydomonas eugametos. This symmetric endonuclease displays unique structural elaborations on its core enzyme fold, and it preferentially cleaves a highly asymmetric target site. This latter property represents an early step, prior to gene fusion, in the generation of asymmetric DNA binding platforms from homodimeric ancestors. The divergence of the sequence, structure, and target recognition behavior of homing endonucleases, as illustrated by this study, leads to the invasion of novel genomic sites by mobile introns during evolution.

Selected figure(s)

Figure 3.
Figure 3. Superpositions of I-CeuI and I-CreI
(A and B) Superposition of I-CeuI is shown in green, and superposition of I-CreI is shown in blue. Superpositions shown from the (A) side and (B) bottom of the enzyme. The LAGLIDADG helices are shown in the same orientations to the right. The rmsd for backbone atoms of individual subunits is vert, similar 2 Å. The relative orientation of the two DNA-contacting β platforms, calculated from the bottom of the conserved LAGLIDADG helices, differs by vert, similar 5° (indicated by a black arrow). This difference is caused by a shift in the packing of the LAGLIDADG helix against the corresponding enzyme core in each subunit (indicated by a red arrow for one subunit), rather than by a rigid body rotation of the two subunits.
(C) Left: Magnification of the superimposed dimer interfaces of I-CeuI and I-CreI, which contain the conserved residues of their respective active sites. Right: The same orientation with only the I-CeuI interface and active sites shown. Catalytic residues of I-CreI are blue, and those of I-CeuI are colored by element type. A single bound calcium ion in the I-CeuI structure is shown; the corresponding anomalous difference density is shown in the right panel. The calcium is bound between the scissile phosphates and the corresponding metal binding residues. The Q93 residue of I-CeuI is modeled from the crystal structure of the Q93R mutant used to solve its structure. Figure 3. Superpositions of I-CeuI and I-CreI(A and B) Superposition of I-CeuI is shown in green, and superposition of I-CreI is shown in blue. Superpositions shown from the (A) side and (B) bottom of the enzyme. The LAGLIDADG helices are shown in the same orientations to the right. The rmsd for backbone atoms of individual subunits is [3]not, vert, similar 2 Å. The relative orientation of the two DNA-contacting β platforms, calculated from the bottom of the conserved LAGLIDADG helices, differs by [4]not, vert, similar 5° (indicated by a black arrow). This difference is caused by a shift in the packing of the LAGLIDADG helix against the corresponding enzyme core in each subunit (indicated by a red arrow for one subunit), rather than by a rigid body rotation of the two subunits.(C) Left: Magnification of the superimposed dimer interfaces of I-CeuI and I-CreI, which contain the conserved residues of their respective active sites. Right: The same orientation with only the I-CeuI interface and active sites shown. Catalytic residues of I-CreI are blue, and those of I-CeuI are colored by element type. A single bound calcium ion in the I-CeuI structure is shown; the corresponding anomalous difference density is shown in the right panel. The calcium is bound between the scissile phosphates and the corresponding metal binding residues. The Q93 residue of I-CeuI is modeled from the crystal structure of the Q93R mutant used to solve its structure.

Figure 5.
Figure 5. DNA Contacts by I-CeuI
(A) The numbering of bases, extending from the center of the four base cleavage site, and the corresponding numbering of the bases in the deposited pdb file. Unambiguous noncovalent contacts to individual bases are shown below. The scissile phosphate groups are red. Base pairs that are conserved between the left and right half-sites, and enzyme residues that are engaged in identical contacts to bases in each half-site, are shaded. Structured water molecules involved in contacts between the DNA target and the enzyme are indicated with circles; the single observed bound metal ion (calcium) is indicated by a circled “M.” This single metal ion is indicated twice in the figure, and it is shared between the scissile phosphates and the enzyme active sites. Residue 93, which is a conserved glutamine in the wild-type enzyme, is present in the structure as a catalytically inactivating arginine (Q93R); this side chain is in contact with the phosphate in each half-site directly 5′ to the scissile phosphate. In structures of I-CreI and I-MsoI, the wild-type glutamine residue participates in coordination of a metal bound water molecule.
(B) Ribbon diagram of the β sheet DNA binding platform and additional elaborations (α-2 and loop 5/6) from I-CeuI; residues participating in DNA-contacts are shown and labeled. The same view of the DNA binding elements of the enzyme with the bound target site is shown below. Figure 5. DNA Contacts by I-CeuI(A) The numbering of bases, extending from the center of the four base cleavage site, and the corresponding numbering of the bases in the deposited pdb file. Unambiguous noncovalent contacts to individual bases are shown below. The scissile phosphate groups are red. Base pairs that are conserved between the left and right half-sites, and enzyme residues that are engaged in identical contacts to bases in each half-site, are shaded. Structured water molecules involved in contacts between the DNA target and the enzyme are indicated with circles; the single observed bound metal ion (calcium) is indicated by a circled “M.” This single metal ion is indicated twice in the figure, and it is shared between the scissile phosphates and the enzyme active sites. Residue 93, which is a conserved glutamine in the wild-type enzyme, is present in the structure as a catalytically inactivating arginine (Q93R); this side chain is in contact with the phosphate in each half-site directly 5′ to the scissile phosphate. In structures of I-CreI and I-MsoI, the wild-type glutamine residue participates in coordination of a metal bound water molecule.(B) Ribbon diagram of the β sheet DNA binding platform and additional elaborations (α-2 and loop 5/6) from I-CeuI; residues participating in DNA-contacts are shown and labeled. The same view of the DNA binding elements of the enzyme with the bound target site is shown below.

The above figures are reprinted by permission from Cell Press: Structure (2006, 14, 869-880) copyright 2006.

Figures were selected by an automated process.