PDBsum entry: 1ght

DNA binding protein

PDB id: 1ght

Contents

Protein chain

105 a.a. *

* Residue conservation analysis

PDB id:

1ght

Name:

DNA binding protein

Title:

Solution structure of the catalytic domain of gamma delta resolvase

Structure:

Transposon gamma-delta resolvase. Chain: a. Fragment: n-terminal catalytic domain. Engineered: yes

Source:

Escherichia coli. Organism_taxid: 562. Expressed in: escherichia coli bl21(de3). Expression_system_taxid: 469008.

NMR struc:

1 models

Authors:

B.Pan,G.P.Mullen

Key ref:

B.Pan et al. (2001). Solution structure of the catalytic domain of gammadelta resolvase. Implications for the mechanism of catalysis. J Mol Biol, 310, 1089-1107. PubMed id: 11501998 DOI: 10.1006/jmbi.2001.4821

Date:

11-Jan-01 Release date: 01-Aug-01

Protein chain

P03012  (TNR1_ECOLI) -  Transposon gamma-delta resolvase

Seq: 183 a.a.

Struc: 105 a.a.

 Gene Ontology (GO) functional annotation 

• Biological process: DNA recombination (1 term)
• Biochemical function: recombinase activity (2 terms)

DOI no: 10.1006/jmbi.2001.4821

J Mol Biol 310:1089-1107 (2001)

PubMed id: 11501998

Solution structure of the catalytic domain of gammadelta resolvase. Implications for the mechanism of catalysis.

B.Pan, M.W.Maciejewski, A.Marintchev, G.P.Mullen.

ABSTRACT

The site-specific DNA recombinase, gammadelta resolvase, from Escherichia coli catalyzes recombination of res site-containing plasmid DNA to two catenated circular DNA products. The catalytic domain (residues 1-105), lacking a C-terminal dimerization interface, has been constructed and the NMR solution structure of the monomer determined. The RMSD of the NMR conformers for residues 2-92 excluding residues 37-45 and 64-73 is 0.41 A for backbone atoms and 0.88 A for all heavy atoms. The NMR solution structure of the monomeric catalytic domain (residues 1-105) was found to be formed by a four-stranded parallel beta-sheet surrounded by three helices. The catalytic domain (residues 1-105), deficient in the C-terminal dimerization domain, was monomeric at high salt concentration, but displayed unexpected dimerization at lower ionic strength. The unique solution dimerization interface at low ionic strength was mapped by NMR. With respect to previous crystal structures of the dimeric catalytic domain (residues 1-140), differences in the average conformation of active-site residues were found at loop 1 containing the catalytic S10 nucleophile, the beta1 strand containing R8, and at loop 3 containing D67, R68 and R71, which are required for catalysis. The active-site loops display high-frequency and conformational backbone dynamics and are less well defined than the secondary structures. In the solution structure, the D67 side-chain is proximal to the S10 side-chain making the D67 carboxylate group a candidate for activation of S10 through general base catalysis. Four conserved Arg residues can function in the activation of the phosphodiester for nucleophilic attack by the S10 hydroxyl group. A mechanism for covalent catalysis by this class of recombinases is proposed that may be related to dimer interface dissociation.

Selected figure(s)

Figure 10.
Figure 10. Active site compari- sons for the NMR solution struc- ture and X-ray structures and a proposed mechanism for covalent catalysis. (a) Comparison of side- chain conformations for S10, D67 and R68 in the solution structure of the resolvase catalytic domain (1- 105) (yellow backbone and thinner, dark-colored side-chains and unprimed labels) and in the dimeric crystal structures (gray backbone and thicker, light-colored side-chains and primed labels). The ten lowest-energy conformations from the NMR structural ensemble are shown. (b) Divergent stereo- view of the conformation of the catalytic side-chains in the mini- mized average structure deter- mined by NMR. (c) The proposed mechanism for covalent catalysis by gd resolvase. In this mechanism, R8, R68 and R71 form charge inter- actions with the scissile phosphate group and promote nucleophilic attack by the catalytic S10 at the phosphodiester. D67 acts as a gen- eral base abstracting the S10 hydroxyl proton, and the S10 hydroxyl group forms a covalent bond to the DNA.

Figure 11.
Figure 11. A structural comparison of gd resolvase (gold and yellow ribbon) to the T5 5 -30exonuclease (cyan back- bone), two enzymes showing no sequence homology. For resolvase, the side-chains for R8, R45, R68, and R71 are shown in blue; the side-chain for D67 is in magenta; and the side-chain for S10 is in green. For the T5 50-3 exonu- clease, the Asp side-chains that coordinate the active-site metal ions are shown in red. The superimposition suggests a similarity of function for the two metal ions coordinated by the Asp residues in the T5 exonuclease and the Arg residues in gd resolvase. R68 and R71 of resolvase are positioned analogously to one of the metal ions (gray spheres), while R8 and R45 are positioned analogously to the second metal ion. Structurally homologous regions were deter- mined using the program DALI 2.0. 30

The above figures are reprinted by permission from Elsevier: J Mol Biol (2001, 310, 1089-1107) copyright 2001.

Figures were selected by an automated process.