Using a forced molecular evolution approach, selecting with mAMSA and DACA, drug resistant mutation G465D has been identified in topoisomerase IIβ on four occasions with two selection agents. This mutation also confers resistance to several other drugs of different classes including AMCA, mAMCA, Etoposide, Ellipticine and Doxorubicin.
The resistance to DACA is very strong at 10- to 20-fold compared with wild-type enzyme. DACA is unlike the other acridines in that its exact mechanism of action is not well understood, with evidence suggesting it acts as a dual topoisomerase I and II poison, so caution must be exercised in interpreting the drug resistance data from the yeast organism. DACA may not be exclusively targeting Topo II (42
An atypical drug resistance phenotype such as that given by βG465D, where resistance is conferred to a range of structurally diverse drugs, implies that the mechanism of resistance is through a reduction in enzyme function giving fewer functional molecules to target as opposed to a decrease in specific interaction with drugs. Mutations of this type have been reported previously in the B′ domain (26
The resistance to mAMSA, the selection agent in three of the four G465D mutations identified, was measured at different time points. The MLC for strains expressing either βG465D or βWT was measured at 6 and 24 h and continuous growth on drug plates was measured after 3–5 days. A clear pattern emerged where the drug resistance decreased over time, from a 10-fold resistance after 6 h exposure, to 2-fold resistance after 24 h exposure, and then after 3–5 days βG465D showed no resistance. These data imply that, while a defect in the enzyme is likely, this defect does not permanently disable any aspect of enzyme function and merely ‘slows’ the turnover.
Indeed, the yeast carrying the mutant plasmid grows at approximately half the rate of yeast with plasmid encoding wild-type Topo IIβ (data not shown). The selection method used ensures that any mutants identified will be functional—if they weren't then the yeast would be unable to grow at the non-permissive temperature, thus the slower growth of the βG465D yeast implies that while it is functional, it is less functional than wild type. If there is less functional topoII target it would give a drug resistance phenotype.
The relaxation shows no significant difference to wild type which is unexpected as an enzyme with lower general function would be expected to have a slower reaction cycle. It is however possible that the experimental set up may be masking a slight impairment of activity. The βG465D decatenation for a single time point showed just 60.3% wild-type activity and subsequent decatenation versus time experiments showed an initial 12-fold rate reduction. Interestingly the βG465D displayed a narrower magnesium optima compared with βWT. In decatenation experiments the MgCl2 concentration was 10 µM, optimal for both βWT and βG465D proteins, and perhaps more importantly the ATP levels were saturating, enough perhaps to compensate for the enzyme's natural deficiency in ATP hydrolysis. ATP dependence assays showed maximum activity at 150 and 50 µM ATP for βG465D and βWT, respectively, and in decatenation assays ATP was at 2 mM.
The reason for this reduced decatenation rate is unclear. It is clear that the ATP requirements for βG465D differ markedly from βWT. Decatenation assays with varying concentrations of ATP showed a 3-fold decrease in ATP affinity with βG465D, and a corresponding 3-fold decrease in ATP hydrolysis activity with this mutant. This is particularly interesting as, while the mutation does lie towards the N-terminal ATPase domain, it is actually located in the B′ domain, close to where the G-helix is predicted to bind (8
Mutations in this region have been reported for human topoIIα and yeast topoII. The topoIIα mutation R450Q (equivalent to βK466), lies adjacent to the mutation identified here (34
). This mutation gives a 2.5-fold resistance to mAMSA and was first identified in the drug resistant leukaemic cell line CEM/VM-1, which was found to have a 2- to 8-fold decrease in ATP hydrolysis activity. In the case of yeast topoII residue G437S, equivalent to βG464, a loss of enzyme stability, reducing the number of functional target molecules, has been reported previously as a reason for drug resistance (45
). The activity profile differed to that of βG465D and αR450Q in that G437S showed drug hypersensitivity and no alteration in ATP requirement and degradation was very rapid. Extrapolating between species can be problematic and has been described previously (33
). Reduced stability is not seen with βG465D, so this is unlikely to be the mechanism of resistance in this case.
The primary sequence of topoIIβ places G465 adjacent to the transducer domain of the N-terminal ATPase domain, which is thought to function by transmitting the ‘information’ of ATP hydrolysis from the N-terminus to the cleavage core through a series of conformational changes. Experiments with WTα have shown that a two-residue insert at position 408, equivalent to βE425 (46
), and a deletion stretching from 350 to 407 (47
) can disrupt communication between domains. In these cases inter-domainal communication was completely abolished, and it is conceivable that a residue contacting this domain may have a communication role. Additional splice variants of human topoIIα have been identified with differently sized inserts in the transducer region of the ATPase domain, at positions K321, Q355 and A401 (48
). It is possible that these alternative forms will have differences in the transduction of conformational changes which could account for differences in activity in vivo
The proximity of the residue to the transducer domain also makes it possible that this inhibits signal transduction through conformational change. The yeast core crystal structure, on which all of the topoII models are based, lacks any data for the region linking the core and ATPase domains as this was too disordered, so it is unclear how these two interact. It is likely however that the ATP hydrolysis activity of the mutant is reduced through inhibition of signal transduction through the transducer domain to the N-terminal region.
The position of G465 on the model based on the yeast structure, plus previous footprinting experiments suggest that glycine 465 lies very close to the area binding the gate helix. Lysines in yeast topoII have been identified that are protected on DNA binding, and one such residue identified was Lys 438. The equivalent residue in WTβ is K466 which is adjacent to G465 (8
). Modelling the G465D mutation onto the yeast core structure using SwissProt shows that while the residue lies in the GyrB homology domain, and would therefore be expected to be involved in ATP hydrolysis, in eukaryotes this is actually located in the B′ domain, on the top surface of the semi-circular groove where the gate helix is thought to bind () (8
). The mutation of residue 465 from a glycine to an aspartic acid doesn't appear to massively change the 3D structure of the enzyme, which is expected as our selection procedure would not select a non-functional mutant. Analysis of this region shows that the mutation gives rise to some localized hydrogen bonding changes that could potentially alter the conformation of the enzyme (). The presence of the negatively charged aspartic acid creates an additional hydrogen bond to the backbone and could create more resistance to conformational change.
Figure 7 Model of human topoIIβ monomer based on the yeast core structure . The effect of the mutation was modelled using Swiss-PdbViewer 3.7, the figures were produced using RasMol. Shown are proteins WTβ in a front view (A) and a side view (more ...)
It is possible that the replacement of a glycine with an aspartic acid, and hence the addition of a negative charge, could alter the charge interactions involved in binding of the gate helix. Fluorescence anisotropy experiments with a 40 bp oligo showed a decreased BMAX value for βG465D, suggesting that the protein is binding in a slightly different conformation giving a consequent change in tumbling rate. However there was no significant difference in KD for the two proteins implying that binding affinity was not effected. This, along with the fact that the oligo used was not physiological, suggests that it is unlikely that impaired DNA binding is responsible for the decrease in decatenation seen with βG465D.
The narrower optimum for MgCl2
is interesting and could perhaps be explained by considering its role in the topoisomerase II mechanism. Mg2+
is thought to be utilized at both the ATP-binding domain and in the cleavage core. In the nucleotide-binding pocket Mg2+
has been shown to contact all of the phosphate groups as well as an invariant Asn residue (23
). In the structure of the N-terminal region of human topoIIα it is also shown to hydrogen bond with a water molecule that is also forming contacts with Glu87, a residue found previously to be a general base ideally positioned to promote nucleophilic attack on the γ-phosphate (50
). It is possible that the altered Mg2+
optima seen could be linked to an impairment in the nucleotide-binding pocket, whereby Glu87 is unable to promote ATP hydrolysis.
βG465 lies within the core region, where Mg2+
is thought to be involved in polarizing the active site tyrosine and stabilizing the cleavable complex intermediate, and is therefore co-ordinated near to the site of gate helix binding. There is also evidence for a structural role for Mg2+
in this region (27
). In this region it is thought that Mg2+
is co-ordinated to a catalytic triad of aspartic acid residues and the active site tyrosine, allowing nucleophilic attack on the phosphate of DNA (40
). The mutation of glycine 465 to aspartic acid introduces a residue that may also co-ordinate Mg2+
. The cleavage site is likely to have a higher affinity for Mg2+
than the D465 region, which would explain why at lower Mg2+
concentrations both proteins are equally active, but at higher Mg2+
levels D465 may also co-ordinate magnesium in an inappropriate way. This additional Mg2+
associated with the protein could either interfere in the phosphoryltransfer reaction directly, or it could impair the movement of domains necessary for enzyme turnover.
The structure of human topoIIα shows that when AMPPNP (an ATP analogue) is bound the transducer domain assumes a packed conformation in which a catalytic lysine residue protrudes into the nucleotide-binding pocket and contacts the γ-phosphate. When ADP is bound however the transducer domain is rotated outward 10°, retracting the lysine from the pocket, in a conformation thought to be necessary for Pi release (49
). It is thought that the Pi release event and removal of the so-called ‘switch lysine’ from the nucleotide-binding pocket may be coupled simultaneously with the separation of the G-segment in the cleavage core (52
). Our data are consistent with this hypothesis, and it is probable that mutation of glycine 465 to aspartic acid impedes the communication between domains mediated by transducer region, giving a reduced ATP hydrolysis rate and slower enzyme turnover.
It is known that the transduction of signal through conformational change is necessary for co-ordination of core and N-terminal domains to give functional enzyme. Here we report a mutation which lies in the core domain, and yet gives a 3-fold reduction in activity at the N-terminal domain implying that transduction of signal has been affected. This mutation confers a resistance to drug that diminishes with time, suggesting that rather than being fatally impeded the enzyme is merely slowed down, a mechanism of resistance which to our knowledge is previously unreported.