PDBsum entry: 2jbg

Hydrolase/inhibitor

PDB id: 2jbg

Contents

Protein chains

87 a.a. *

121 a.a. *

Ligands

SO4 ×2

Metals

_ZN ×2

Waters ×316

* Residue conservation analysis

PDB id:

2jbg

Name:

Hydrolase/inhibitor

Title:

Crystal structure of the mutant n560a of the nuclease domain of cole7 in complex with im7

Structure:

Colicin-e7 immunity protein. Chain: a, c. Synonym: colicin e7 immunity protein, imme7, microcin-e7 immunity protein. Engineered: yes. Colicin e7. Chain: b, d. Fragment: nuclease domain, residues 446-576. Synonym: colicin-e7.

Source:

Escherichia coli. Organism_taxid: 316407. Strain: w3110. Expressed in: escherichia coli. Expression_system_taxid: 562.

Resolution:

2.20Å R-factor: 0.202 R-free: 0.249

Authors:

H.Huang,H.S.Yuan

Key ref:

H.Huang and H.S.Yuan (2007). The conserved asparagine in the HNH motif serves an important structural role in metal finger endonucleases. J Mol Biol, 368, 812-821. PubMed id: 17368670 DOI: 10.1016/j.jmb.2007.02.044

Date:

07-Dec-06 Release date: 03-Apr-07

Protein chains

Q03708  (IMM7_ECOLX) -  Colicin-E7 immunity protein

Seq: 87 a.a.

Struc: 87 a.a.

Protein chains

Q47112  (CEA7_ECOLX) -  Colicin-E7

Seq: 576 a.a.

Struc: 121 a.a.*

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

 Gene Ontology (GO) functional annotation 

• Biological process: cytolysis (4 terms)
• Biochemical function: protein binding (4 terms)

DOI no: 10.1016/j.jmb.2007.02.044

J Mol Biol 368:812-821 (2007)

PubMed id: 17368670

The conserved asparagine in the HNH motif serves an important structural role in metal finger endonucleases.

H.Huang, H.S.Yuan.

ABSTRACT

The HNH motif is a small nucleic acid binding and cleavage module, widespread in metal finger endonucleases in all life kingdoms. Here we studied a non-specific endonuclease, the nuclease domain of ColE7 (N-ColE7), to decipher the role of the conserved asparagine and histidine residues in the HNH motif. We found, using fluorescence resonance energy transfer (FRET) assays, that the DNA hydrolysis activity of H545 N-ColE7 mutants was completely abolished while activities of N560 and H573 mutants varied from 6.9% to 83.2% of the wild-type activity. The crystal structures of three N-ColE7 mutants in complex with the inhibitor Im7, N560A-Im7, N560D-Im7 and H573A-Im7, were determined at a resolution of 1.9 A to 2.2 A. H573 is responsible for metal ion binding in the wild-type protein, as the zinc ion is still partially associated in the structure of H573A, suggesting that H573 plays a supportive role in metal binding. Both N560A and N560D contain a disordered loop in the HNH motif due to the disruption of the hydrogen bond network surrounding the side-chain of residue 560, and as a result, the imidazole ring of the general base residue H545 is tilted slightly and the scissile phosphate is shifted, leading to the large reductions in hydrolysis activities. These results suggest that the highly conserved asparagine in the HNH motif, in general, plays a structural role in constraining the loop in the metal finger structure and keeping the general base histidine and scissile phosphate in the correct position for DNA hydrolysis.

Selected figure(s)

Figure 4.
Figure 4. The omit difference (F[o]-F[c]) maps of N560A–Im7, N560D–Im7 and H573A–Im7 at the mutation site. (a) The N560A map contoured at 3σ shows the short side-chain density of A560. (b) In N560D, the side-chain of D560 swings out of the loop and points outwards (map contoured at 3σ). (c) In H573A, the electron density for the mutated residue A573 is not seen in the difference map (contoured at 3σ). A zinc ion is located in the active site and binds to H544, H569 and a water molecule.

Figure 6.
Figure 6. Stereo views of the superposition of the HNH motif in the wild-type N-ColE7 (in grey) and N560D mutant (in pink). Only the backbone atoms in the HNH motif were used for the least-squares fitting. Half of the loop is disordered in the N560D mutant, and as a result, the side-chain of H545 is tilted and the phosphate ion bound in the active site is shifted. The distance between H545 N^δ1 atom to the phosphate oxygen atom changes from 2.41 Å in the wild-type enzyme to 3.72 Å in the N560D mutant. This result indicates that the replacement of N560 in the HNH motif disturbs the loop structure and in turn changes the orientation of the imidazole ring of the general base histidine (H545), which is responsible for the reduced endonuclease activity.

The above figures are reprinted by permission from Elsevier: J Mol Biol (2007, 368, 812-821) copyright 2007.

Figures were selected by the author.