PDBsum entry 1v14

Hydrolase

PDB id: 1v14

Contents

Protein chains

132 a.a. *

DNA/RNA

Metals

_MG ×6

Waters ×33

* Residue conservation analysis

References listed in PDB file

Key reference

Title

Structure-Based analysis of the metal-Dependent mechanism of h-N-H endonucleases.

Authors

M.J.Maté, C.Kleanthous.

Ref.

J Biol Chem, 2004, 279, 34763-34769. [DOI no: 10.1074/jbc.M403719200]

PubMed id

15190054

Abstract

Controversy surrounds the metal-dependent mechanism of H-N-H endonucleases, enzymes involved in a variety of biological functions, including intron homing and DNA repair. To address this issue we determined the crystal structures for complexes of the H-N-H motif containing bacterial toxin colicin E9 with Zn(2+), Zn(2+).DNA, and Mg(2+).DNA. The structures show that the rigid V-shaped architecture of the active site does not undergo any major conformational changes on binding to the minor groove of DNA and that the same interactions are made to the nucleic acid regardless of which metal ion is bound to the enzyme. The scissile phosphate contacts the single metal ion of the motif through distortion of the DNA brought about by the insertion of the Arg-96-Glu-100 salt bridge into the minor groove and a network of contacts to the DNA phosphate backbone that straddle the metal site. The Mg(2+)-bound structure reveals an unusual coordination scheme involving two H-N-H histidine residues, His-102 and His-127. The mechanism of DNA cleavage is likely related to that of other single metal ion-dependent endonucleases, such as I-PpoI and Vvn, although in these enzymes the single alkaline earth metal ion is coordinated by oxygen-bearing amino acids. The structures also provide a rationale as to why H-N-H endonucleases are inactive in the presence of Zn(2+) but active with other transition metal ions such as Ni(2+). This is because of coordination of the Zn(2+) ion through a third histidine, His-131. "Active" transition metal ions are those that bind more weakly to the H-N-H motif because of the disengagement of His-131, which we suggest allows a water molecule to complete the catalytic cycle.

Figure 3.
FIG. 3. A hydrogen-bonding network connects DNA distortion to approach of the scissile bond to the H-N-H motif metal center. Stereo representation of the main hydrogen-bonding interactions between the E9 DNase and the DNA minor groove in the Zn2+ complex, with identical interactions observed in the Mg2+-bound complex. The DNA and protein residues are cream and green, respectively, the Zn2+ ion is magenta, and hydrogen bonds are plotted as dotted lines. The scissile bond of the DNA can only approach the metal ion if the DNA is distorted. This is accomplished by the insertion of the Arg-96-Glu-100 salt bridge into the minor groove and stabilization of this configuration through a number of hydrogen-bonding interactions to the DNA phosphates through residues Arg-5, Asp-51, and Arg-54.

Figure 4.
FIG. 4. Simulated annealing omit maps around the active site for the complexes of H103A E9 DNase·dsDNA bound to Zn2+ (A) and Mg2+ (B). The metal ion, the scissile phosphate, and histidines 102, 127, and 131 were omitted for the calculation, and the resulting maps were plotted at a contour level of 2.0 . The coordination of both metal ions is indicated with dotted lines. The figure highlights how the H-N-H motif is an adaptable metal binding center. It is able to accommodate both the tetrahedral coordination chemistry of a Zn2+ ion (A) and then, through subtle reorientations of the H-N-H histidine residues and the inclusion of DNA phosphodiester oxygen atoms and a water molecule, the octahedral geometry required for Mg2+ ion binding (B). Only five of the possible six coordination sites are discernible for the Mg2+ ion at the current level of resolution.

The above figures are reprinted by permission from the ASBMB: J Biol Chem (2004, 279, 34763-34769) copyright 2004.

Secondary reference #1

Title

Specificity in protein-Protein interactions: the structural basis for dual recognition in endonuclease colicin-Immunity protein complexes.

Authors

U.C.Kühlmann, A.J.Pommer, G.R.Moore, R.James, C.Kleanthous.

Ref.

J Mol Biol, 2000, 301, 1163-1178. [DOI no: 10.1006/jmbi.2000.3945]

PubMed id

10966813

Figure 4.
Figure 4. Hydrogen bonding interactions at the E9 DNase-Im9 interface. (a) and (b) show similar orientations of the interface and are stereo representations in which Im9 is coloured yellow with light side-chains and the DNase red with dark side-chains. Details are given in Table 2 and Table 3. (a) Direct hydrogen bonds between Im9 and the E9 DNase surrounding the core of the interface, made up of a stacking interaction between Tyr54 Im9 with Phe86 E9 DNase. (b) Water-mediated hydrogen bonds.

Figure 10.
Figure 10. Comparison of hydrogen bonding interactions to conserved water molecules in the E7 DNase-Im7 (from [Ko et al 1999]), dark shading, and E9 DNase-Im9 complexes (present work), light shading. With the exception of Asn90 (which is glutamine in the E7 DNase), conserved side-chains and backbone atoms are involved in coordinating the interfacial water molecules. Hydrogen bonds and side-chain numbering are for the E9 DNase-Im9 complex.

The above figures are reproduced from the cited reference with permission from Elsevier