PDBsum entry: 1rr8

Isomerase/DNA

PDB id: 1rr8

Contents

Protein chain

494 a.a. *

DNA/RNA

Ligands

TTG-TTC

Waters ×304

* Residue conservation analysis

PDB id:

1rr8

Name:

Isomerase/DNA

Title:

Structural mechanisms of camptothecin resistance by mutations in human topoisomerase i

Structure:

5'- d( Ap Ap Ap Ap Ap Gp Ap Cp Tp T Gp Gp Ap Ap Ap Ap Ap Tp Tp Tp Tp T)-3'. Chain: a. Engineered: yes. 5'- d( Ap Ap Ap Ap Ap Tp Tp Tp Tp Tp Cp Cp Ap Ap Gp Tp Cp Tp Tp Tp Tp T)-3'. Chain: b.

Source:

Synthetic: yes. Homo sapiens. Human. Organism_taxid: 9606. Gene: top1. Expressed in: spodoptera frugiperda. Expression_system_taxid: 7108. Expression_system_cell_line: sf9.

Biol. unit:

Trimer (from PQS)

Resolution:

2.60Å R-factor: 0.242 R-free: 0.294

Authors:

J.E.Chrencik,B.L.Staker,A.B.Burgin,L.Stewart,M.R.Redinbo

Key ref:

J.E.Chrencik et al. (2004). Mechanisms of camptothecin resistance by human topoisomerase I mutations. J Mol Biol, 339, 773-784. PubMed id: 15165849 DOI: 10.1016/j.jmb.2004.03.077

Date:

08-Dec-03 Release date: 06-Jul-04

Protein chain

P11387  (TOP1_HUMAN) -  DNA topoisomerase 1

Seq: 765 a.a.

Struc: 494 a.a.*

* PDB and UniProt seqs differ at 4 residue positions (black crosses)

Enzyme reactions

• Enzyme class: E.C.5.99.1.2 - Dna topoisomerase.
• Reaction: ATP-independent breakage of single-stranded DNA, followed by passage and rejoining.

 Gene Ontology (GO) functional annotation 

• Cellular component: chromosome (1 term)
• Biological process: DNA topological change (2 terms)
• Biochemical function: DNA binding (3 terms)

DOI no: 10.1016/j.jmb.2004.03.077

J Mol Biol 339:773-784 (2004)

PubMed id: 15165849

Mechanisms of camptothecin resistance by human topoisomerase I mutations.

J.E.Chrencik, B.L.Staker, A.B.Burgin, P.Pourquier, Y.Pommier, L.Stewart, M.R.Redinbo.

ABSTRACT

Human topoisomerase I relaxes superhelical tension associated with DNA replication, transcription and recombination by reversibly nicking one strand of duplex DNA and forming a covalent 3'-phosphotyrosine linkage. This enzyme is the sole target of the camptothecin family of anticancer compounds, which acts by stabilizing the covalent protein-DNA complex and enhancing apoptosis through blocking the advancement of replication forks. Mutations that impart resistance to camptothecin have been identified in several regions of human topoisomerase I. We present the crystal structures of two camptothecin-resistant forms of human topoisomerase I (Phe361Ser at 2.6A resolution and Asn722Ser at 2.3A resolution) in ternary complexes with DNA and topotecan (Hycamtin), a camptothecin analogue currently in widespread clinical use. While the alteration of Asn722 to Ser leads to the elimination of a water-mediated contact between the enzyme and topotecan, we were surprised to find that a well-ordered water molecule replaces the hydrophobic phenylalanine side-chain in the Phe361Ser structure. We further consider camptothecin-resistant mutations at seven additional sites in human topoisomerase I and present structural evidence explaining their possible impact on drug binding. These results advance our understanding of the mechanism of cell poisoning by camptothecin and suggest specific modifications to the drug that may improve efficacy.

Selected figure(s)

Figure 1.
Figure 1. (a) A 2.3 Å resolution crystal structure of human topoisomerase I in covalent complex with a 22 base-pair duplex DNA containing topotecan and an Asn722Ser mutation. The CAP region sits above the DNA and is composed of core sub-domains I and II. The CAT region sits below the DNA and consists of core sub-domain III and the C-terminal domain. The linker domain, which follows in primary sequence the CAP and connects the CAP and CAT regions of the enzyme, protrudes away from the body of the protein structure. CPT-resistant mutations discussed here (Table 1) are indicated in CPK and colored cyan. (b) Molecular surface of human topoisomerase I in the ternary complex of the enzyme, DNA and topotecan (TTC; magenta), viewed in roughly the same orientation as in (a) A channel vert, similar 16 Å in width at the interface of the CAP and CAT regions appears to allow TTC to gain access to the active site in the protein–DNA complex.

Figure 2.
Figure 2. (a) Electron density (2|F[obs]|−|F[calc]|, 2.3 Å resolution, contoured at 1σ) showing the Asn722Ser mutation in the structure of the mutant human topoisomerase I–DNA–TTC ternary complex. (b) Electron density (2|F[obs]|−|F[calc]|, 2.6 Å resolution, contoured at 1σ) showing the Phe361Ser mutation in the structure of the mutant human topoisomerase I–DNA–TTC ternary complex. (c) Stereo view of the electron density for topotecan as observed in the 2.3 Å resolution crystal structure of the Asn722Ser mutant human topoisomerase I in ternary complex with DNA and the drug (2|F[obs]|−|F[calc]|, contoured at 1σ).

The above figures are reprinted by permission from Elsevier: J Mol Biol (2004, 339, 773-784) copyright 2004.

Figures were selected by the author.