PDBsum entry 1nop

Hydrolase/DNA

PDB id: 1nop

Contents

Protein chain

425 a.a. *

DNA/RNA

Ligands

_DT

LYS-LEU-ASN-TYR-LEU

VO4 ×2

Waters ×96

* Residue conservation analysis

References listed in PDB file

Key reference

Title

Crystal structure of a transition state mimic for tdp1 assembled from vanadate, Dna, And a topoisomerase i-Derived peptide.

Authors

D.R.Davies, H.Interthal, J.J.Champoux, W.G.Hol.

Ref.

Chem Biol, 2003, 10, 139-147. [DOI no: 10.1016/S1074-5521(03)00021-8]

PubMed id

12618186

Abstract

Tyrosyl-DNA phosphodiesterase (Tdp1) is a member of the phospholipase D superfamily and acts as a DNA repair enzyme that removes stalled topoisomerase I- DNA complexes by hydrolyzing the bond between a tyrosine side chain and a DNA 3' phosphate. Despite the complexity of the substrate of this phosphodiesterase, vanadate succeeded in linking human Tdp1, a tyrosine-containing peptide, and a single-stranded DNA oligonucleotide into a quaternary complex that mimics the transition state for the first step of the catalytic reaction. The conformation of the bound substrate mimic gives compelling evidence that the topoisomerase I-DNA complex must undergo extensive modification prior to cleavage by Tdp1. The structure also illustrates that the use of vanadate as the central moiety in high-order complexes has the potential to be a general method for capturing protein-substrate interactions for phosphoryl transfer enzymes, even when the substrates are large, complicated, and unusual.

Figure 3.
Figure 3. Hydrogen Bonding Contacts between Tdp1 and the Vanadate-Peptide-DNA Substrate Transition State AnalogTdp1, peptide, and DNA are colored as in Figure 1A, with the vanadate moiety in green and hydrogen bonds indicated by dashed lines. Residues 232–242 of Tdp1 have been omitted for clarity. Hydrogen bonds to the vanadate moiety are displayed in (A), hydrogen bonds to the peptide moiety are displayed in (B), and hydrogen bonds to the DNA moiety in (C).

Figure 4.
Figure 4. Electrostatic Potential Surface of Tdp1The molecular surface is colored between −10kT (red) and +10kT (blue) and was generated with the program GRASP [35]. The orientation of the Tdp1 structure is the same as in Figure 1A. The peptide-vanadate-DNA substrate mimic is displayed as a stick structure. The yellow V indicates the position of the vanadate residue in the active site. The DNA moiety extends above the active site, bound in the narrow, positively charged half of the substrate binding groove. The peptide moiety is located below the active site in a relatively neutral portion of the wider substrate binding cleft.

The above figures are reprinted by permission from Cell Press: Chem Biol (2003, 10, 139-147) copyright 2003.

Secondary reference #1

Title

The crystal structure of human tyrosyl-Dna phosphodiesterase, Tdp1.

Authors

D.R.Davies, H.Interthal, J.J.Champoux, W.G.Hol.

Ref.

Structure, 2002, 10, 237-248. [DOI no: 10.1016/S0969-2126(02)00707-4]

PubMed id

11839309

Figure 3.
Figure 3. Schematic View of the Hydrogen Bonding Pattern Observed in the Active Site of Tdp1Bonds of residues from the N-terminal domain of Tdp1 are colored blue, and residues from the C-terminal domain are colored yellow. This view was created with the program LIGPLOT [38].

The above figure is reproduced from the cited reference with permission from Cell Press

Secondary reference #2

Title

Insights into substrate binding and catalytic mechanism of human tyrosyl-Dna phosphodiesterase (tdp1) from vanadate and tungstate-Inhibited structures.

Authors

D.R.Davies, H.Interthal, J.J.Champoux, W.G.Hol.

Ref.

J Mol Biol, 2002, 324, 917-932. [DOI no: 10.1016/S0022-2836(02)01154-3]

PubMed id

12470949

Figure 6.
Figure 6. Schematic representation of the hydrogen bonding pattern observed in the active site of Tdp1-tungstate. Coloration is the same as in Figure 4.

Figure 9.
Figure 9. Proposed reaction mechanism for human Tdp1. (a) Specific binding of the Tdp1 substrate positions the phosphate moiety in the center of the active site, with non-bridging oxygens interacting with the amino groups of Lys265 and Lys495. (b) His263 performs the first nucleophilic attack of the reaction mechanism. A trigonal bipyramidal transition state is formed and the apical tyrosine moiety is the leaving group. His493 acts as a general acid, donating a proton to the leaving group. (c) After completion of the first step of the catalytic reaction, a phosphohistidine intermediate remains. (d) The second nucleophilic attack of the reaction mechanism is carried out by a water molecule activated by His493. This step proceeds through a trigonal bipyramidal transition state similar to the one in the first catalytic step. (e) The active site of Tdp1 is regenerated, and the other product, 3'-phosphate DNA, is released.

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