Type IIA Topoisomerase
Created by Taylor Powell
Type IIA Topoisomerase is a cytoplasmic enzyme that regulates the topological state of DNA. All cells have a fundamental need for Topoisomerase as replication of DNA can create knots and interlocking rings in the strands of nucleotides that must be resolved before division of the chromosomes (11). Further, the double-stranded helical structure of DNA will cause the nucleotides to contort and supercoil, a processes that must be reversed (11). Topoisomerases overcome these fundamental cellular problems by cleaving, manipulating, and then religating DNA strands (11).
The general mechanism of all Type II Topoisomerases is described by a protein clamp model (8). First, the tyrosyl group of DNA backbone nucleophilically attacks the enzyme. Simultaneously, the DNA strand breaks, and a phosphotyrosine bond is formed between the Human IIA Topoisomerase enzyme and the DNA (8). The result of this reaction is a covalent attachment of the protein to the 5’ end of DNA (5). At this point, the broken DNA segment, which is called the gated (or G-) segment, is open for a second region of DNA, called the transported (or T-) segment, to pass through the open gate (8). ATP-mediated closure of the enzyme jaws drive this process (8). In this manner, Topoisomerase II is able to mediate the topological state of DNA. To function as described, all topoisomerases require both ATP hydrolysis and magnesium(II) metal ions (5).
The crystallographic structure of the Homo Sapiens Type IIA DNA Topoisomerase in complex with phosphoaminophosphonic acid-adenylate ester (HT2ATPase•AMPPNP) was solved to 1.86Å by researchers at Harvard University (PDB ID = 1ZXM) (9). The structure contains two substraits. The prosthetic group, AMPPNP, is a non-hydrolyzable ATP analog. It produces changes in overall conformation when compared to the enzyme•ADP complex. The magnesium(II) metal ion, Mg2+, is also in the crystal complex (9). This ion is crucial in coordinating the protein with AMPPNP in the ATP binding pocket. All three phosphates of the AMPPNP are coordinated to the γ-carbonyl oxygen of Asn-91, and two water molecules complete the metal coordination shell of Mg2+ (9). One of these water molecules is then hydrogen-bonded to Glu-87. It is thought that Glu-87 activates an attacking water molecule, but the structure was inconclusive (9).
The portion of human Topoisomerase IIA that was crystalized has a molecular weight of 91177.19 Da and an isoelectric point of 8.99 (12). The enzyme is 75Å wide and 79Å long (9). It is 400 amino acid residues in length, and it is composed a secondary structure consisting of 28% α-helixes, 28% β-strands, and 44% random coil (9). The enzyme is a heart-shaped dimer composed of two identical protomers.
Two discrete modules are present on the crystallized protein. The N-terminal module binds nucleotides while the C-terminal module communicates conformational changes associated with the binding and hydrolysis of ATP.
TheN-terminal module of human Type IIA Topoisomerase contains an eight-stranded β-sheet backed by four α-helices. This is the characteristic fold of the GHKL superfamily of nucleotide-binding enzymes (so named for founding members DNA gyrase, Hsp90, histidine kinase, and MutL) (7,9). Traditionally, this structure is referred to as a 5Y-CAP domain (11). It contains a winged-helix with a catalytic tyrosine that becomes covalently bonded to the 5’ end of the DNA after nucleophilic attack (11). Further, a highly conserved arginine residue near the catalytic tyrosine is thought to help position and stabilize the reaction transition state (11).
The C-terminal module of human Type IIA Topoisomerase contains a four-stranded β-sheet with an α-helix on one side and two on the other. This module is known as the transducer because it communicates conformational information associated with the binding and hydrolysis of ATP between the ATPase domain and the rest of the protein (9). This module is also known as the Toprim domain (so named for its founding members Topoisomerase and Primase) (11).
Each monomer of the dimeric human Type IIA Topoisomerase contains one ATP-binding pocket. In the crystallographic structure this pocket is filled with the non-hydrolyzable ATP analog AMPPNP, which is held in place by a number of hydrogen bonds to the protein. The adenine ring is held in place by hydrogen bonding to the Asn-120 side chain carbonyl and by water-mediated hydrogen-bonding to Asn-120, Thr-215, and Tyr-34. The ribose ring of AMPPNP is stabilized though hydrogen bonding to Ser-149 and Asn-150. The backbone amide groups from Arg-162, Asn-163, Gly-164, Tyr-165, Gly-166, and Ala-167 contact the α- and γ-phosphates. The α-phosphate is further restrained by hydrogen bonds to Lys-168 and Asn-91 Finally, the β-phosphate is held by hydrogen bonds to Ser-148 (9). The Lys-378 residue is believed to stabilize the transition state of the hydrolysis of ATP. It forms a salt bridge with the γ-phosphate of AMPPNP (9).
An alternate conformation to the HT2ATPase•AMPPNP complex is the HT2ATPase•ADP complex. This complex was solved to a resolution of 2.51Å by the same crystallographers at Harvard University (9). Like the AMPPNP complex, HT2ATPase•ADP is a pseudosymmetric dimer; however, as their superimposition suggests, greater variation in the crystal structure of HT2ATPase•APD creates a less constrained overall conformation. Structures of the key sites are very similar. The Mg2+ ion and the ADP are in essentially the same positions. The ATP binding pocket makes the same set of direct and water-mediated hydrogen bonds to ADP as AMPPNP, except that the β-phosphate has an additional hydrogen bond to Asn-150. This additional bond is due to the loss of the γ-phosphate in ADP (9).
In the HT2ATPase•ADP complex, the only residue that undergoes a major change in orientation is Lys-378. Lys-378 is a highly conserved residue in all Type II Topoisomerases, and it forms a salt bridge with the γ-phosphate in the HT2ATPase•AMPPNP complex. When the γ- phosphate is hydrolyzed, the rigid-body movement of the protein retracts Lys-378. For this reason, it is believed that Lys-378 acts as both a sensor and relay for the nucleotide-bound status of the enzyme (9).
Two proteins of similar form and function to human DNA Topoisomerase IIA include the ParE subunit of DNA Topoisomerase IV from Escherichia coli (PDB ID = 1S16) and DNA Gyrase B from Thermus thermophilus (PDB ID = 1KIJ). These proteins were chosen for comparison because they share both form and function with the protein of interest, human Topoisomerase IIA. The function of all three proteins is to mediate the topological state of DNA in the cytoplasm and nucleus of the cell. Further, both E. coli Topoisomerase IV and T. thermophilus Gyrase B show primary and tertiary structural similarities to human HT2ATPase.
Like human HT2ATPase, E. coli Topoisomerase IV (PDB ID = 1S16) is responsible for decatenating daughter chromosomes following genome replication (2). The results of DALI (Z=54.3, Ideal = 63.6) and protein BLAST (E=2e-14) indicate that the ParE subunit of this enzyme shows both primary and tertiary similarities to HT2ATPase (3, 6). The two proteins show an 88% convergence of primary structure (6). As with HT2ATPase, the ParE subunit contains the ATPase site associated with the GHKL superfamily of phosphotransferases in the N-terminal domain and a C-terminal transducer domain (2). Topoisomerase IV is a pseudosymmetric, heat-shaped dimer that forms a hollow cavity representative of the protein clamp. Further, the enzyme is associated with the same ligands as HT2ATPase, AMPPNP and Mg2+.
DNA Gyrase B from T. thermophilus (PDB ID = 1KIJ) catalyzes the negative supercoiling of DNA (1). Meaning, this enzyme is capable of maintaining the genomes of bacteria slightly under-wound (11). Again, this is an example of an enzyme maintaining the topological state of DNA though hydrolysis of ATP. The results of DALI (Z=27.8, Ideal = 63.6) and protein BLAST (E=6e-25) indicate that this enzyme shows both primary and tertiary similarities to HT2ATPase (3, 6). The two proteins show a 94% convergence of primary structures (6). The quaternary structure of Gyrase is an A2B2 tetramer (1); however, the AB dimer form of the protein shows significant similarities to the HT2ATPase dimer. Again, a heartshaped pseudosymmetric structure with a cavity at one end is the result of dimerization. However, unlike HT2ATPase and E. coli Topoisomerase IV, the ligands in complex with the protein are different. DNA Gyrase B is in complex with novobiocin, a powerful inhibitor of the enzyme (1).
Human Topoisomerases have been of recent research interest due to their role as possible anticancer drug targets. The function of Topoisomerases are critical for a cell’s viability. Therefore, if the Topoisomerase can be inactivated in target cells, they will die.
There are two classes of Topoisomerase-targeting anticancer drugs. Class I drugs stabilize the covalently bonded Topoisomerase-DNA catalytic intermediates (4). By stabilizing the protein•DNA complex, the DNA break will lead to cell death (11). These drugs are referred to as “Toposiomerase poisons” because they turn the enzyme into a deadly cellular toxin (4). Class II Topoisomerase-targeting anticancer drugs interfere with the catalytic function of the enzyme. Because these drugs block the function of Topoisomerase without stabilizing the covalent protein•DNA complex, they are referred to as “Topoisomerase inhibiters” (4).
Type II DNA Topoisomerases are specifically targeted by two varieties of drugs including the intercalators and the non-intercalators. In general, both of these varieties act on cancer cells by interfering with Topoisomerase II reactions in the cell cycle at S phase or during the segmentation of the chromosomes during mitosis. Intercalators disturb the position of the 3-OH end of the DNA relative to the DNA 5’•Topoisomerase II covalent complex. This permutation leads to breaks in DNA strands or misalignment of DNA when the two strands are religated by the enzyme (4). It has been found that these drugs are effective against different forms of leukemia and breast cancer (4).
Nonintercalators are drugs composed of two domains. One domain binds DNA while the other binds proteins. These drugs work by interacting with the protein portion of the DNA 5’•Topoisomerase II covalent complex. The result is a potent mitotic inhibitor that is active against testicular cancer, malignant lymphoma, and central nervous system tumors, among others (4). Unfortunately, none of the topoisomerase IIA-drug complexes have yet to by crystalized. However, the crystal structure of a Type IIβ Topoisomerase in complex with the promising anticancer drug, etoposide, has recently been solved (10).
Human Topoisomerase IIβ in complex with DNA and the anti-cancer drug etoposide (PDB DI = 3QX3) shows that the drug-bound protein assumes a more open quaternary conformation than in the DNA•protein structures that have been previously solved (10). The most significant changes are seen in the domains that usually make hydrogen-bonded contact to the DNA (10). In this manner, the drug prevents religating of cleaved DNA segments by separating key catalytic residues (10). Therefore, etoposide is a class I “Topoisomerase poison” (4).