Molecular mechanism of the camptothecin resistance of Glu710Gly topoisomerase IB mutant analyzed in vitro and in silico

The mutations (Leu617Ile, Arg621His and Glu710Gly) found in the CPT-11 resistant HCT116 clones by Gongora et al.[24] have been introduced in the single copy yeast plasmid (YCp) expressing hTopI under the GAL1 promoter. To assess the in vivo consequences of mutants expression, the viability and CPT sensitivity of Top1Δ yeast cells (EKY3), transformed with GAL1-hTop1 constructs, have been tested. The expression vector carries the Ura-selectable marker and is maintained by selection in synthetic complete SC Ura-medium. At least five independent clones were selected from each transformation. Serial dilutions of yeast cells, transformed with the indicated plasmids, have been spotted on plates containing dextrose or galactose, supplemented with different CPT concentrations, to assess drug sensitivity following the yeast growth. The data show that yeast cells expressing the wild type protein exhibit a deficiency in viability in the presence of 10 ng/ml CPT, while the three mutations render the enzyme resistant to CPT (Figure 1A). However, the mutants display a different sensitivity to CPT concentration: Arg621His and Glu710Gly produce viable colonies until 100 ng/ml of CPT while the Leu617Ile is less resistant growing only up to 50 ng/ml.

The effect of 60 μM CPT on the ability of wild type and Glu710Gly enzymes to mediate relaxation of supercoiled plasmid, under standard assay conditions, has been assessed incubating 18 ng of each enzyme with 0.5 μg of a negative supercoiled plasmid in a time course kinetics from 0.25 to 30 minutes. Since CPT is dissolved in dimethyl sulfoxide (DMSO), an assay of the enzyme activity in the presence of an identical amount of DMSO without CPT has been also carried out, to show that DMSO does not affect the relaxation activity of the enzyme. The reactions have been stopped and the products resolved by agarose gel electrophoresis. As shown in Figure 1B, the two enzymes exhibit comparable relaxation activities, since they completely relax the supercoiled DNA after 0.5 minutes of incubation (Figure 1B, lanes 2). The presence of CPT considerably slows down the relaxation of native hTop1, a full relaxation being observed only after about 8 minutes from the drug addiction (Figure 1B, lane17). On the other hand the Glu710Gly mutant completely relaxes the supercoiled DNA in 1–2 min in presence of the drug (Figure 1B lane 13–14), confirming its CPT resistance. The same experiment has been carried out using a more diluted enzyme, confirming that the relaxation rate for the wild type and mutant is comparable and that the mutant is CPT resistant (data not shown). The resistance of the Glu710Gly has been analyzed in detail following the different steps of the catalytic cycle and studying its structural and dynamical properties by MD simulation.

The time course of the cleavage of the wild type and Glu710Gly mutant has been followed using a suicide cleavage substrate. In detail, a 5′-end radiolabeled oligonucleotide CL14 (5′-GAAAAAAGACTTAG-3′) has been annealed to the CP25 (5′-TAAAAATTTTTCTAAGTCTTTTTTC-3′) complementary strand, to produce a duplex with an 11-base 5′-single-strand extension. The enzyme preferentially cuts at the site indicated by the arrow, as shown in top of Figure 2A. Using this substrate the religation step is almost totally precluded, because the AG-3′ is too short to be religated, leaving the enzyme covalently attached to the 12 oligonucleotide 3′-end [17]. For a suicide substrate incubated with an excess of wild type or mutant enzyme, the cleaved DNA fragments have been resolved in a time course experiment in a denaturing polyacrylamide gel as reported in Figure 2A. The amount of fragment, normalized to the maximum value of the wild type protein, plotted as a function of time in Figure 2B, suggests that both enzymes have an almost identical cleavage rate (k_cl), being the initial part of the curves almost identical in both reactions. The two enzymes however don’t reach the same plateau value leaving us with the hypothesis that a small amount of AG-3′ could be religated and that the mutant could religate this short oligonucleotide faster than the wild type enzyme. In order to address this hypothesis a different cleavage substrate has been used to follow the rate of cleavage. In detail, a 5′-end radiolabeled oligonucleotide, named CL14-U (5′-GAAAAAAGACTUAG-3′), having the deoxyribo-thymine (dT) in position 12 substituted with a ribo-Uracil (rU), has been annealed to the CP25 (5′-TAAAAATTTTTCTAAGTCTTTTTTC-3′) complementary strand, to produce a duplex with an 11-base 5′-single-strand extension. With this substrate, when the enzyme cuts at the preferential site indicated by an arrow at the top of Figure 3A, the 2′-OH of the ribose attacks the 3′-phosphotyrosyl linkage between the enzyme and ribonucleotide, releasing hTop1 and leaving a 2′,3′-cyclic phosphate end [25]. In this way the enzyme is not anymore covalently linked to the substrate and the short oligonucleotide cannot be religated. The wild type and the mutant have been incubated with the substrate and the fragments have been resolved in a time course experiment in a denaturing polyacrylamide gel and the result is reported in Figure 3A. The cleaved oligo (CL1 in Figure 3A) runs like a 12 mer since no protein is attached to it and the plot of the amount of this fragment as a function of time, normalized to the maximum value of the wild type protein, unambiguously demonstrates that the wild type protein and the Glu710Gly mutant have the same rate of cleavage and reach the same plateau value (Figure 3B).

The DNA religation step has been studied by testing the ability of both enzymes to religate the oligonucleotide R11 (5′-AGAAAAATTTT-3′) in the presence or absence of CPT, added to the cleavage complex obtained incubating the suicide substrate with an excess of enzyme. Aliquots have been removed at different times, the reaction stopped by addition of SDS and the products analyzed by polyacrylamide gel electrophoresis (Figure 4A). The percentage of the remaining covalent complex (CL1), normalized to the value at t=0 is plotted, as a function of time, in Figure 4B. The data show that the Glu710Gly mutant has a higher religation rate when compared with the wild type enzyme (Figure 4A, compare lanes 2–6 with lanes 11–15). The presence of CPT strongly decreases the religation rate in the wild type protein (lanes 7–10), whereas has only a small effect for the Glu710Gly mutant (lanes 16–19) bringing the religation rate to a value similar to the one observed for the wild type in absence of the drug, as shown by the quantitative plot of Figure 4B.

Root mean square displacement (RMSD) plots along the 75 ns simulation of the native and mutated proteins show that in both proteins the core is stable (see Additional file 1) and that the largest deviation is due to the linker domain. This is confirmed by the root mean square fluctuations (RMSF), where the larger contribution comes from the linker domain (Figure 5). The two systems show comparable values along the sequence, except for the peaks at 466–471 and 608–610 that have higher values for the mutant and the 494–497 that have higher values in the wild type.

In the X-ray structures [5, 15, 26] helix 19 (residues 679–710, one of the two helices of the linker domain), directly faces helix 17 (residues 612–629), through two salt bridges (Glu710-Arg621 and Asp707-Arg624) permitting the communication between the linker domain and the cap-core structure of the protein (Figure 6A). In the wild type simulation the side chains of these residues keep forming a network of salt bridges for a large percentage of time (85% and 96%) as shown in Figure 6C, left side, where a representative snapshot of the simulation is shown. In the simulation of the Glu710Gly mutant, besides the obvious disappearance of the Glu710-Arg621 salt bridge, the Asp707-Arg624 salt bridge is also broken (only 9% existence), thus separating the communication between the base of the linker with the core of the protein (Figure 6C, right side). Another effect of the mutation is observed in the bundle of helices (16, 17 and 21) neighbouring the mutation zone, (see Figure 6A), where helices 16 and 21 change their relative orientation since Trp732, located on helix 21, interacts with Thr605 in the wild type, but with Leu606 in the mutant simulation as shown by two representative snapshots of the simulations in Figure 6B. As a consequence the C-terminal of helix 16, downstream of this interaction increases its flexibility explaining the peak seen in the RMSF for residues 607–610 (Figure 5).

The Glu710Gly mutation has an important effect on the concerted motions of the protein as can be noted comparing the dynamic cross-correlation matrix of the C-alpha atoms of the protein residues during the trajectories of the wild type and the mutant (Figure 7, top and bottom halves respectively). In the figure the pairs of Cα atoms characterized by strong correlated motions are represented by a pixel of red, yellow or green color, while anticorrelated motions are shown in cyan, blue and violet. The mutant is endowed with a much lower degree of correlation. In detail, in the wild type, the linker has a strong anticorrelated motion with the C-teminal domain and the C-terminal region of subdomain III (squares in Figure 7) and a lower, but still pronounced, anticorrelated motion with the nose cone helices (representing the protein region that contacts the DNA on the opposite side respect to the linker, downstream of the cleavage site, circled in Figure 7). In the mutant these anticorrelated motions are lost or strongly weakened (Figure 7 bottom). A similar loss of correlation has been found for the Thr729Lys that also displays CPT resistance [18].

DMSO and CPT were purchased from Sigma-Aldrich. CPT was dissolved in 99.9% DMSO to a final concentration of 4 mg/ml (11.5 mM) and stored at −20°C.

ANTI-FLAG M2 monoclonal affinity gel, FLAG peptide and ANTI-FLAG M2 monoclonal antibody were purchased from Sigma-Aldrich.

Saccharomyces cerevisiae top1 null strain EKY3 (ura3-52, his3Δ200, leu2Δ1, trp1Δ63, top1:TRP1, MATα) previously described [31] was used to express the hTop1 gene. YCpGAL1-e-hTop1 single copy plasmid was described previously [31, 32]. Leu617Ile, Arg621His and Glu710Gly were generated by oligonucleotide-directed mutagenesis of the YCpGAL1-wild type in which the human topoisomerase I is expressed under the galactose inducible promoter in a single-copy plasmid. The epitope-tagged construct YCpGAL1-e-wild type contains the N-terminal sequence FLAG: DYKDDDY (indicated with ‘e’), recognized by the M2 monoclonal antibody. The epitope-tag was subcloned into YCpGAL1-hTop1Leu617Ile, YCpGAL1-hTop1Arg621His and YCpGAL1-hTop1Glu710Gly to produce the YCpGAL1-e-hTop1Leu617Ile, YCpGAL1-e-hTop1Arg621His and YCpGAL1-e-hTop1Glu710Gly construct. The cloning reactions were transformed into XL10-Gold E. coli cells (Agilent Technologies) and positive clones were identified by sequencing the extracted plasmid DNA.

Yeast EKY3 strains were transformed with YCp50, YCpGAL1-e-hTop1, YCpGAL1-e-hTop1Leu617Ile, YCpGAL1-e-hTop1Arg621His and YCpGAL1-e-hTop1Glu710Gly vectors by LiOAc treatment [33] and selected on synthetic complete (SC)-uracil medium supplemented with 2% dextrose. Transformants were grown to an A⁵⁹⁵=0.3 and 5 μl aliquots of serial 10-fold dilutions were spotted onto SC-uracil plates plus 2% dextrose or 2% galactose, with or without the indicated concentrations of CPT.

EKY3 yeast cells, transformed with the YCpGAL1-e-hTop1 and YCpGAL1-e-hTop1Glu710Gly were grown overnight on SC-uracil plus 2% dextrose, at an optical density of A⁵⁹⁵=1.0 they were diluted 1:100 in SC-uracil plus 2% raffinose. At an optical density of A⁵⁹⁵=1.0, the cells were induced with 2% galactose for 6 h. Cells were then centrifuged, washed with cold water and resuspended in 2 ml buffer/g cells (50 mM Tris–HCl, pH 7.4, 1 mM EDTA, 1 mM EGTA, 10% glycerol and protease inhibitors cocktail from Roche, supplemented with 0.1 mg/ml sodium bisulfate, 0.8 mg/ml sodium fluoride, 1mM Phenylmethanesulfonylfluoride (PMSF) and 1mM DTT). After addition of 0.5 volumes of 425–600 μm diameter glass beads, the cells were disrupted by vortexing for 30 seconds alternating with 30 seconds on ice and then were centrifuged at 15000g for 30 minutes. For homogenous protein preparations, the whole extracts were applied to an ANTI-FLAG M2 affinity gel (Sigma-Aldrich) already equilibrated in according with the manufacturer protocol. Then, columns were washed with 20 volumes of TBS (50 mM Tris–HCl pH 7.4 and 150 mM KCl) supplemented with the protease inhibitors, prior to load the lysate. Elution of e-hTop1, or e-hTop1Glu710Gly, was performed by competition with five column volumes of a solution containing 1mg of FLAG peptide (DTKDDDDK) in TBS. Fractions of 500 μl were collected and 40% glycerol was added in all preparations, which were stored at −20°C [17]. Protein levels and integrity were assessed by immunoblot with the monoclonal anti M2 antibody (Sigma-Aldrich). The hTop1 and hTop1Glu710Gly similar concentrated fractions were also compared to the purified hTop1 with a known concentration (provided from Topogene) by immunoblot using the ab58313 Anti-Top1 antibody (Abcam) and ab97240 goat polyclonal Secondary Antibody (Abcam). The relative concentration of the two chosen fractions was estimated by a densitometry quantification using ImageJ software [34]. The in vitro experiments have been performed using equal amount of purified hTop1 and hTop1Glu710Gly.

The activity of 1 μl of hTop1 (16ng/μl) or hTop1Glu710Gly (16ng/μl) was assayed in 30 μl of reaction volume containing 0.5 μg of negatively supercoiled pBlue-Script KSII(+) DNA, that is present in both dimeric and monomeric forms and reaction buffer (20 mM Tris–HCl pH 7.5, 0.1 mM Na₂EDTA, 10 mM MgCl₂, 5 μg/ml acetylated bovine serum albumin and 150 mM KCl).

The effect of CPT on enzyme activity was measured by adding DMSO or 100μM of the drug to the reactions, that were stopped with 0.5% SDS after each time-course point at 37°C. The samples were resolved in a 1% (w/v) agarose gel in 48 mM Tris, 45.5 mM boric acid, 1 mM EDTA at 10 V/cm. The gels were stained with ethidium bromide (0.5 μg/ml), destained with water and photographed using a UV transilluminator.

Oligonucleotide CL14 (5′-GAAAAAAGACTTAG-3′) that contain a hTop1 high affinity cleavage site, was 5′-end labelled with [γ³²P] ATP. The CP25 complementary strand (5′-TAAAAATTTTTCTAAGTCTTTTTTC-3′) was 5′-end phosphorylated with unlabeled ATP. The two strands were annealed with a 2-fold molar excess of CP25 over CL14 [35]. The suicide cleavage reactions were carried out by incubating 20 nM of the duplex DNA with an excess of hTop1 or hTop1Glu710Gly enzymes at 25°C, in 20 mM Tris–HCl pH 7.5, 0.1 mM Na₂EDTA, 10 mM MgCl₂, 5 μg/ml acetylated BSA, and 150 mM KCl, in a final volume of 50 μl [36]. At various time points 5 μl aliquots were removed and the reaction stopped with 0.5% (w/v) SDS. After ethanol precipitation samples were resuspended in 5 μl of 1 mg/ml trypsin and incubated at 37°C for 60 minutes. However a short trypsin resistant peptide is always left explaining why the CL1 migrates slower than the CL14 oligonucleotide [20]. Samples have been analyzed by denaturing 7 M urea/20% polyacrylamide gel electrophoresis in TBE (48 mM Tris, 45.5 mM Boric Acid, 1 mM EDTA).

Oligonucleotide CL14-U (5′-GAAAAAAGACTUAG-3′) was 5′ end labelled and annealed with CP25 as for the CL14. 20 nM substrate has been incubated with an excess of hTop1 or hTop1Glu710Gly enzymes in 20 mM Tris–HCl pH 7.5, 0.1 mM Na₂EDTA, 10 mM MgCl₂, 5 μg/ml acetylated BSA, 150 mM KCl, at 25°C in a final volume of 40 μl. At various time points 5 μl aliquots were removed and the reaction stopped with 0.5% (w/v) SDS and directly loaded without ethanol precipitation and trypsin digestion. Samples have been analyzed by denaturing 7 M urea/20% polyacrylamide gel electrophoresis in TBE (48 mM Tris, 45.5 mM Boric Acid, 1 mM EDTA). In both experiments the percentage of cleaved substrate (CL1) was determined by PhosphorImager and ImageQuant software and normalized on the total amount of radioactivity in each lane.

20 nM of CL14/CP25 (radiolabeled as previously described in the cleavage kinetic experiment) was incubated with an excess of hTop1 or hTop1Glu710Gly for 60 minutes at 25°C followed by 30 minutes at 37°C in 20 mM Tris–HCl pH 7.5, 0.1 mM Na₂EDTA, 10 mM MgCl₂, 50 μg/ml acetylated BSA, and 150 mM KCl. After the formation of the cleavage complex (CL1) a 5μl aliquote was removed and used as time 0 point, then DMSO or 100 μM CPT were added and religation reaction was started by adding a 200-fold molar excess of R11 oligonucleotide (5′-AGAAAAATTTT-3′) over the CL14/CP25 [20]. 5 μl aliquots were removed at various time points, and the reaction stopped with 0.5% SDS. After ethanol precipitation, samples were resuspended in 5 μl of 1 mg/ml trypsin and incubated at 37°C for 60 minutes. Samples were analyzed by denaturing 7 M urea/20% polyacrylamide gel electrophoresis in 48 mM Tris, 45.5 mM Boric Acid, 1 mM EDTA. The percentage of remaining cleavage complex was quantified by ImageQuant software, normalized to the total radioactivity for each lane and to the value at t=0 and finally plotted as a function of time.

The initial configuration of the wild type hTop1, in covalent complex with a 22 base pair linear double helix DNA substrate, has been modeled from the crystallographic structures (PDB 1K4S and 1TL8, respectively) as already reported [23].

The Glu710Gly mutant was generated using the rotamer module presents in the Chimera package [37]. The systems have been modeled using the AMBER03 all-atom force field [38] implemented by Sorin and Pande [39] in the GROMACS MD package version 4.5.4 [40]. The proteins was placed in a rhombic dodecahedron box with a minimum distance of 14 Å from the box edges, then filled with water molecules described by means of the TIP3P rigid potential and Na+ counter-ions [41] were added to neutralize DNA-enzyme total charge via the genion tool of the GROMACS package. The resulting WT system is composed of 9456 protein atoms, 1400 DNA atoms, 58919 water molecules, 20 Na+ ions, for a total of 187633 atoms.

Particle Mesh Ewald method (PME) was used to handle long-range electrostatic interactions using a cutoff of 1.2 nm in real space and the same cutoff value was used for Van der Waals interactions [42]. The LINCS algorithm was used to constrain bond lengths and angles [43]. Relaxation of solvent molecules and Na+ ions was initially performed keeping solute atoms restrained to their initial positions, in three cycles with decreasing force constant of 1000, 600 and 300 kJ/(mol N nm), for 300 ps each. The two systems have then been simulated for 75 ns with a time step of 2.0 fs and the neighbor list was updated every 10 steps. Temperature was kept constant at 300 K using the velocity rescale Berendsen method with a coupling constant of 0.1 ps during sampling, while pressure was kept constant at 1 bar using the Parrinello-Rahman barostat with a coupling constant of 1.0 ps during sampling [44].

Standard analyses, as root mean square deviations (RMSD) and fluctuations (RMSF), hydrogen bonds, distances evaluations etc. were calculated using tools of GROMACS MD 4.5.3 [40] package. Correlation maps were calculated on the C-alpha atoms as described [18] with in-house written codes. Images were produced with VMD [45] and Chimera [37].