Type II Topoisomerase - Structure of Type IIA Topoisomerases

Structure of Type IIA Topoisomerases

Type IIA topoisomerases consist of several key motifs: an N-terminal GHKL ATPase domain (for gyrase, Hsp, kinase and MutL), a Toprim domain (sometimes called a Rossmann fold), which exists in both type II topoisomerases, type IA topoisomerases, and bacterial primase (DnaG), a central DNA-binding core (which structurally forms a heart-shaped structure), and a variable C-terminal domain.

Eukaryotic type II topoisomerases are homodimers (A₂), while prokaryotic type II topoisomerases are heterodimers (A₂B₂). Prokaryotes have the ATPase domain and the Toprim fold on one polypeptide, while the DNA cleavage core and the CTD lies on a second polypeptide. For gyrase, the first polypeptide is called GyrB and the second polypeptide is called GyrA. For topo IV, the first polypeptide is called ParE and the second polypeptide is called ParC.

The structures of the N-terminal ATPase domain of gyrase (Wigley, Davies, Dodson, Maxwell, and Dodson, Nature 1991) and yeast topoisomerase II (Classen and Berger, Proceedings of the National Academy of Science, 2003, PDB ID=1PVG) have been solved in complex with AMPPNP (an ATP analogue), showing that two ATPase domains dimerize to form a closed conformation. For gyrase, the structure has a substantial hole in the middle, which is presumed to accommodate the T-segment.

Linking the ATPase domain to the Toprim fold is a helical element known as the transducer domain. This domain is thought to communicate the nucleotide state of the ATPase domain to the rest of the protein. Modifications to this domain affect topoisomerase activity, and structural work done by the Verdine group shows that the ATP state affects the orientation of the transducer domain (Journal of Biological Chemistry, 2006).

The central core of the protein contains a Toprim fold and a DNA-binding core that contains a winged helix domain (WHD), often referred to as a CAP domain, since it was first identified to resemble the WHD of catabolite activator protein. The catalytic tyrosine lies on this WHD. The Toprim fold is a Rossmann fold that contains three invariant acidic residues that coordinate magnesium ions involved in DNA cleavage and DNA religation (Avarind, Leipe, Konin, Nucleic Acids Research 1998). The structure of the Toprim fold and DNA-binding core of yeast topoisomerase II was first solved by Berger and Wang (Nature 1996, PDB ID = 1BGW), and the first gyrase DNA-binding core was solved by Morais Cabral et al. (Nature 1997, PDB ID = 1AB4). The structure solved by Berger revealed important insights into the function of the enzyme. The DNA-binding core consists of the WHD, which leads to a tower domain. A coiled-coil region leads to a C-terminal domain that forms the main dimer interface for this crystal state (often termed the C-gate). While the original topoisomerase II structure shows a situation where the WHDs are separated by a large distance, the structure of gyrase shows a closed conformation, where the WHD close.

The topoisomerase II core was later solved in new conformations, including one by Fass et al. (Nature Structure Biology 1999, PDB ID = 1BJT) and one by Dong et al. (Nature 2007, PDB ID = 2RGR). The Fass structure shows that the Toprim domain is flexible and that this flexibility can allow the Toprim domain to coordinate with the WHD to form a competent cleavage complex. This was eventually substantiated by the Dong et al. structure that was solved in the presence of DNA. This last structure showed that the Toprim domain and the WHD formed a cleavage complex very similar to that of the type IA topoisomerases and indicated how DNA-binding and cleavage could be uncoupled, and the structure showed that DNA was bent by ~150 degrees through an invariant isoleucine (in topoisomerase II it is I833 and in gyrase it is I172). This mechanism of bending resembles closely that of integration host factor (IHF) and HU, two architectural proteins in bacteria. In addition, while the previous structures of the DNA-binding core had the C-gate closed, this structure captured the gate open, a key step in the two-gate mechanism (see below).

More recently, several structures of the DNA-bound structure have been solved in an attempt to understand both the chemical mechanism for DNA cleavage and the structural basis for inhibition of topoisomerase by antibacterial poisons.

The C-terminal region of the prokaryotic topoisomerases has been solved for multiple species. The first structure of a C-terminal domain of gyrase was solved by Corbett et al. (Proceedings of the National Academy of Science, 2004, PDB ID = 1SUU), and the C-terminal domain of topoisomerase IV was solved by Corbett et al. (Journal of Molecular Biology, 2006, PDB ID = 1zvt and 1zvu). The structures formed a novel beta barrel, which bends DNA by wrapping the nucleic acid around itself. The bending of DNA by gyrase has been proposed as a key mechanism in the ability of gyrase to introduce negative supercoils into the DNA. This is consistent with footprinting data that shows that gyrase has a 140-base-pair footprint. Both gyrase and topoisomerase IV CTDs bend DNA, but only gyrase introduces negative supercoils.

Unlike the function of the C-terminal domain of prokaryotic topoisomerases, the function of the C-terminal region of eukaryotic topoisomerase II is still not clear. Studies have suggested that this region is regulated by phosphorylation and this modulates topoisomerase activity, however more research needs to be done to investigate this.