by Cameron Kubota
DNA gyrase belongs to a class of enzymes called DNA topoisomerases, the class of DNA enzymes that are responsible for aiding the winding and unwinding of DNA supercoiled helices. Supercoiled helices occur when DNA strands are wound too tightly (positive supercoils) or not wound tightly enough (negative supercoils). Topoisomerases received its name as it only temporarily changes the topology of the DNA, resulting in a chemical isomer of the wound or unwound DNA strands. DNA gyrase is particularly unique because it is the only topoisomerase that can both relax positively supercoiled DNA and introduce negative supercoils into the DNA (most topoisomerases only relieve the positively supercoiled DNA).1) In 1976, Gellert and his coworkers were attempting to establish Escherichia coli host factors required for bacteriophage wavelength site-specific integration when they discovered an ATP-dependent enzyme that was capable of introducing negative supercoils into chains of DNA, a discovery that changed the way we look at the unwinding and replication of DNA today.2) DNA gyrase is vital in this replication cycle of DNA in bacteria and other prokaryotes, such as the E. Coli sample Gellert was working with, but are only found in a single species of eukaryotes. This species of eukaryotes is called Plasmodium falciparum, the protozoan parasite that is the main culprit for malaria in humans. Because of this property, gyrase has become a targeted enzyme for antibacterial research. By deactivating the gyrase, the bacteria will die off, and because humans don’t have DNA gyrase, shutting off the gyrase will have no effect on our own cells.3) Although humans don't have DNA gyrase, another enzyme called topoisomerase II is found in human cells to relieve twisting stress in DNA replication.
In a 1978 study, it was found that gyrase contains two subunits; one smaller subunit that is directly responsible for interacting with ATP and a larger subunit that, due to its similar migration to another protein on sodium dodecyl sulfate/polyacrylamide gel electrophoresis, was determined to be similar to that of the NalA protein, a protein that had previously been extracted from E. Coli cells.4) These subunits are called GyrA (the larger subunit) and GyrB (the smaller subunit) and both contain domains significant to the introduction of negative supercoils into the DNA helix.
GyrA is a dimer subunit that is responsible for interacting directly with only the DNA strand and not ATP. In 2007, Raghu Ram and his team of scientists studied the structure of GyrA and found that there was extensive fold conservation exhibited between the GyrA subunit of E.Coli and of Plasmodium falciparum (PfGyrase). They found that the Tyrosine 122 (Y122) residue in particular was exactly the same throughout all species that contain DNA gyrase. They concluded that this residue is critical to the function of the enzyme, because it directly interacts with the broken 5'-end of each DNA strand. Another segment that saw complete conservation was the quinolone resistant region between residues 67-106.5) Another region that is important to the function of gyrase is the CAP region (residues 71-140) in GyrA, a region that strongly resembles the DNA binding region in E.Coli catabolite activator protein. This region must be conserved because it plays an integral part in binding to the DNA T-segment, allowing the gyrase to introduce negative supercoils in the DNA helix.6)
In 2007, a team of scientists examined the DNA gyrase of Escherichia Coli and were able to report the structural model of the GyrB subunit at ~12-15 Å resolution. They found that the dimer GyrB subunit, or the subunit responsible for interacting with ATP, is composed of three subdomains, the ATPase, Toprim, and Tail.7) These three domains take the shape of a tadpole, with the ATPase forming the head, the Tail subdomain forming the tadpole's tail, and the Toprim connecting the head and tail. Scientists use the term “tadpole” in order to better visualize the structure of the GyrB subunit. From observations between both E.Coli gyrase and Plasmodium falciparum gyrase, Raghu Ram and his team of scientists found that there are two major sections comprised of 50 residues, called disordered loops, that are not conserved, but all eight crucial residues are.8) Although there are huge differences between the gyrase from E.Coli and that of the parasite, the overall structure and function is conserved.
The ATPase subdomain consists of the residue chain from the Serine 22 residue to the Threonine 392 residue (S2 to T392), nearly half of the entire GyrB subunit. This structure contains an 8-strand β-sheet and multiple α-helices that creates a pocket in which ATP readily binds in order to drive the enzymatic uncoiling of the DNA strands. The amino-terminus is found at the head of the “tadpole”, leaving a continuous density for the following domains. The carboxyl-terminus is found in an α-helix that can adopt different conformations depending on the situation and is referred to as a “transducer” because it acts as both a sensor for the interaction with the DNA substrate and as a mediator that links the binding of the DNA with ATP hydrolysis to regulate the enzymatic activity of the gyrase.9)
The Toprim is the subdomain that is found on the elbow connecting the ATPase to the Tail subdomain, or the head to the tail of the tadpole, and consists of residues R393 to P533. The structure of this subdomain is not well understood, but it was found that the α/β fold is structurally conserved across all type IA and type II topoisomerases.10) Using the most complete crystallographic model of the Toprim, it was found that there is a central β-sheet surrounded by α-helices that forms a compact fold. The Toprim subdomain coordinates with Mg2+ ions and is vital to the cleavage-relegation reaction of the DNA strands the DNA gyrase performs.11) Due to the outward orientation of the subdomain, the Toprim subdomain is able to interact with the active site located on GyrA.
The Tail subdomain is referred to as the tail of the tadpole and consists of residues L534 to I804. Like the Toprim subdomain, the Tail has no well-known crystallographic structure. However, Costenaro and his team were able to determine a very likely structure of this subdomain. They found that the Tail is composed of two sections called Tail-1 and Tail-2. Tail-1 lies closest to the Toprim fold and due to its long, flexible structure, will adopt a different conformation to optimize the function of the Tail subdomain. Tail-2 is mostly helical and is found farthest from the ATPase subdomain. Tail-2 greatly interacts with GyrA and the DNA strand and although the function of the Tail is not well established, team Costenaro believe that the Tail plays an important role in the interaction with and introduction of negative supercoils in the DNA double helix.12)
DNA gyrase is an important enzyme found in species that have circular strands of DNA. When the helicase enzyme separates the annealed DNA helix, due to the cyclic nature of the DNA, supercoiling occurs (stressed, “over” coiling of the DNA strands). DNA gyrase is a unique type II topoisomerase because it is the only one that can introduce negative supercoils into the structure of DNA to relieve the strain from the supercoiling and allow the helicase to continue splitting the DNA strands. Some characteristic mechanisms and reactions that DNA gyrase can undergo are:
DNA must first bind to the gyrase in order for any reaction and catalysis to take place. Approximately 120 base pairs of the DNA wraps around the gyrase protein in a positive superhelical fashion and forms a DNA-gyrase complex. To alleviate some of the resulting stress added to the DNA helix from the positive superhelical wrapping, negative superhelical turns are introduced in another portion of the DNA helix. The portion of the DNA wrapped around the gyrase is then cleaved and the 5' ends of both strands interact with the GyrA and become covalently attached through phosphotyrosine residues. The 3' ends of both strands do not covalently attach to the gyrase. Instead, they are held in place noncovalently, either through forces maintaining the integrity of the DNA helix, interactions with the 3' hydroxyl group and the gyrase, or the interactions of the wrapped helix and the gyrase. By securing the cleaved DNA strand to the gyrase, this will ensure that the DNA helix will not unravel in order to relieve the negative supercoil twist. Once secured, the DNA helix is passed through the gap formed at the break site attached to the gyrase and the broken ends of the strands are reattached, using two ATP molecules to do so (the ATP molecules interact with ATPase on GyrB and are cleaved to form ADP and inorganic phosphate, the resulting energy used to drive the reaction). The re-bonding of the DNA strands on the gyrase introduce three negative twists into the the DNA helix and one positive twist, resulting in three negative supercoils in total.14)
While the overall tertiary and even secondary structures of both PfGyrase (Plasmodium falciparum gyrase) and EcGyrase (Escherichia Coli gyrase) are nearly identical, there are many differences in the primary structure that allows them to function and adapt to their respective environments. Comparing PfGyrA to EcGyrA, PfGyrA shows a 57% homology (57% of the residues are similar in chemical properties) at the N-terminus and 45% homology at the C-terminus but is only 38% identical (38% are EXACTLY identical) at the N-terminus and 24% identical at the C-terminus. This means that although many residues are important to conserve completely across species, more than half of the other residues are species-determinant, whether it shows as being partially conserved or completely unconserved. PfGyrB also shows key differences when compared to EcGyrB. PfGyrB shows 41% homology and 28% identity when compared to EcGyrB, meaning that more than half of the residues are not even partially conserved across the species (The percentage of homology and identity are greatly decreased from GyrA comparison due to the 50 residue loops in PfGyrB that show no conservation at all). Furthermore, both PfGyrA and PfGyrB contain N-terminus extensions of about 120-160 extra amino acids that demonstrate no structural conservation to the EcGyrase units.15) Although structurally PfGyrase and EcGyrase show major differences, both function in the same manner as all residues that are important to introducing negative supercoils in the DNA helix are at least homologous if not identical.
Although Plasmodium falciparum is the only eukaryote that has DNA gyrase to relax the positive supercoils and introduce negative supercoils, other eukaryotes have enzymes that carryout similar functions, so while DNA gyrase is not found in human cells, human topoisomerase II is needed to relieve torsional stress in human cells, as the unwinding of DNA strands at one point in the DNA helix must be caused by overwinding in a different part of the DNA helix, even if the DNA strand is not cyclical like that of Plasmodium falciparum.16)
The two distinct isoforms of topoisomerase II that are found in human cells are referred to as topoisomerase IIα and topoisomerase IIβ. Most structural studies of topoisomerase II (Top2) have been done on yeast, but for most eukaryotes, the residues are conserved. In Top2, there are two distinct domains that play integral parts in DNA replication function, the ATPase domain and breakage-reunion domain.
The N-terminal domain of the protein, the bottom region in Figure 6, carries the portion that is responsible for binding to high-energy ATP molecules and for cleaving the high-energy bond to use the resulting energy to function. Like DNA gyrase, Top2 breakage-domain consists of a central Toprim region which is followed by the winged helix domain that carries the active site residue, tyrosine. Interestingly, the winged helix domain is similar to that of the CAP region found in DNA gyrase. The C-terminal domain shows little to no conservation across species and is not even conserved across Top2α and Top2β. However, this residue is particularly important as it is required for nuclear localization, regulation of enzyme activity by post-translational modification, and regulation of enzyme function through protein:protein interactions.17)
The ATPase region, the top region in Figure 6, consists of a GHKL fold in the N-terminal region and a transducer in the C-terminal region. The transducer domain signals ATP binding to the breakage reunion domain by actually shifting its position upon ATP binding that also triggers other conformational changes in the breakage reunion domain as well. The transducer region is the most important domain in the ATPase unit as it supplies the residues needed to interact with the ATP molecule.18)
It was found that the main function of the human topoisomerase IIα is to solve the problem of overwinding in DNA replication and not the topoisomerase IIβ. DNA replication generates positive supercoils in front of the replication fork, so without the presence of Top2α, DNA helicase would not be able to continue splitting the DNA strands. Should the superhelical coils persist, it is possible that the DNA at the replication fork can isomerize by migration of the positive supercoiling into wrapping of the two replicated strands, a structure that is referred to as a precatenane. In these cases, Top2 is needed to relieve the precatenane structure while Top1 is used to relieve the superhelical twists in the DNA helix.19)