Pyridoxal 5'-phosphate inactivates DNA topoisomerase IB by modifying t
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《核酸研究医学期刊》
1 Biochimie des Signaux Régulateurs Cellulaires et Moléculaires, Université Pierre et Marie Curie, CNRS FRE 2621, 96 Boulevard Raspail, 75006 Paris, France, 2 Département de Biologie et Pharmacologie Structurales, ENS Cachan, CNRS UMR 8113, 61 Avenue du Président Wilson, 94235 Cachan cedex, France, 3 Accelrys, 20 Rue Jean Rostand, 91898 Orsay cedex, France, 4 Institute of Molecular Biology, National Academy Science of Armenia, 7 Hasratyan Street, Yerevan, Armenia and 5 CNRS UMR 8126, Institut Gustave Roussy, 94805 Villejuif cedex, France
* To whom correspondence should be addressed. Tel: +331 53 63 40 76; Fax: +331 53 63 40 77; Email: dergara@ccr.jussieu.fr
ABSTRACT
The present results demonstrate that pyridoxal, pyridoxal 5'-phosphate (PLP) and pyridoxal 5'-diphospho-5'-adenosine (PLP-AMP) inhibit Candida guilliermondii and human DNA topoisomerases I in forming an aldimine with the -amino group of an active site lysine. PLP acts as a competitive inhibitor of C.guilliermondii topoisomerase I (Ki = 40 μM) that blocks the cleavable complex formation. Chemical reduction of PLP-treated enzyme reveals incorporation of 1 mol of PLP per mol of protein. The limited trypsic proteolysis releases a 17 residue peptide bearing a lysine-bound PLP (KPPNTVIFDFLGK*DSIR). Targeted lysine (K*) in C.guilliermondii topoisomerase I corresponds to that found in topoisomerase I of Homo sapiens (K532), Candida albicans (K468), Saccharomyces cerevisiae (K458) and Schizosaccharomyces pombe (K505). In the human enzyme, K532, belonging to the active site acts as a general acid catalyst and is therefore essential for activity. The spatial orientation of K532–PLP within the active site was approached by molecular modeling using available crystallographic data. The PLP moiety was found at close proximity of several active residues. PLP could be involved in the cellular control of topoisomerases IB. It constitutes an efficient tool to explore topoisomerase IB dynamics during catalysis and is also a lead for new drugs that trap the lysine general acid.
INTRODUCTION
The DNA topoisomerases I and II are key enzymes that control the DNA topological states during DNA replication, transcription, repair, recombination and chromatin remodeling. Eukaryotic topoisomerases IA and IB catalyze topological rearrangements of DNA through cleavage and religation of one DNA strand (1–4), either by passing the unbroken strand through the break (topoisomerases IA) (5) or by rotating the broken strand around the unbroken strand (topoisomerases IB) (6,7). The sequential mechanism of the strand rotation controlled by eukaryotic topoisomerases IB involves the reversible formation of a 3'-O-phosphoro-tyrosine bond between the active site tyrosine and the cleaved DNA (8). DNA cleavage and religation can be separated using partial DNA duplexes (9–11). The anti-tumoral derivatives of camptothecin, including the water soluble topotecan used for the treatment of ovarian and colon carcinoma (12,13), stall topoisomerases IB on the DNA between the cleavage and religation steps (14) by intercalation into the active-site-bound DNA (15).
The crystal structure of human topoisomerase I non-covalently bound to DNA reveals that the bilobed enzyme completely wraps around its DNA cognate (7,16–18) with a vast majority of the contacts occurring with the phosphate backbone. Only the base-specific interaction concerns the -amino group of the conserved K532 and the base O2 atom of the consensual thymine on the scissile strand, upstream from the cleavage site (position –1). This interaction has been proposed to stabilize a transition state required for the strand alignment during the religation step (19). The conserved catalytic Y723 and the positively charged residues R488, R590 and H632 occupy positions near the scissile phosphate group so that Y723 can attack the scissile phosphate group, while R488, R590 and H632 stabilize the transition state of the scissile phosphate, and K532 (equivalent to vaccinia K167) acts as the general acid catalyst (20,21). The enzyme conformation that results from binding to DNA allows Y723 to join the active site (22,23). The DNA controlled rotation of topoisomerase IB requires specific concerted motions (24), but these are hindered by intercalation of topotecan into the active site-bound DNA (15). Its global processivity is greatly enhanced in vitro by weak anions and magnesium (25). A concerted mechanism of proton relay has been proven essential in vaccinia topoisomerase I during the transesterification steps that control the DNA strand break, involving the residues K167, R130 (the latter being equivalent to human R488) and the DNA 5' oxygen of the scissile phosphate (26). The crucial role of a lysine residue side chain in this proton relay has been recently confirmed with the K532 residue of human topoisomerase IB (19). Protonation of the DNA 5' oxygen facilitates the nucleophilic attack of the scissile phosphate by Y723.
Topoisomerase IB is a recognized target for anti-cancer drugs (27,28). Camptothecin represents the prototype of a large class of genotoxic drugs that act as topoisomerase IB poisons, while a second class, acting as topoisomerase I suppressors, consists in catalytic inhibitors that compete with DNA to bind to the enzyme. For such drugs, the relationships between the enzyme inactivation and the anti-proliferative effects are still intriguing as in the presence of a topoisomerase I poison they usually prevent the cleavable complex formation whereas, alone, they exhibit genotoxic properties in cultured cells compound (29)].
The vitamin B6-related pyridoxal 5'-phosphate (PLP) is a lysine-binding coenzyme for a number of enzymes involved in amino acid metabolism, including aminotransferases (30). It also binds to enzymes that recognize phosphate-containing substrates or effectors, allowing its use to probe critical lysine residues within the dNTP-binding domain of DNA polymerases (31–34), RNA polymerases (35–37) and HIV reverse transcriptase (38). We have previously reported that PLP acts as an efficient inhibitor of the C.guilliermondii topoisomerase I (39). Using PLP, pyridoxal (PL) and pyridoxal 5'-diphospho-5'-adenosine (PLP-AMP), we now confirm the critical catalytic function of a lysine residue in C.guilliermondii topoisomerase I, equivalent to residue K532 of the human topoisomerase IB active site (20,21). We demonstrate that PLP is a catalytic inhibitor that competes with the binding of the enzyme to DNA in forming a Schiff base with a lysine residue of the active site. PLP that has been already shown in supra-physiological amount to possess anti-proliferative (40) and anti-tumoral (41) activities could therefore belong to the class of topoisomerase IB inhibitors. Molecular modeling based on the active site of the human topoisomerase I crystal structure (42) provides some information on the spatial organization of PLP adducted on the critical lysine.
MATERIALS AND METHODS
Purification of topoisomerase I from C.guilliermondii (25)
Unless otherwise specified, the purification was carried out at 4°C. C.guilliermondii var. membranaefaciens was cultured as described previously (43). The C.guilliermondii cells were suspended in a buffer containing 50 mM Tris–HCl, pH 7.4, 10 mM KCl, 1 mM EDTA, 1 mM DTT (TK10ED) plus 0.1 M sucrose and lysed in a Dyno Mill disrupter (Willy A. Bachofen AG Maschinenfabrik, Basel, Switzerland) at 3000 r.p.m. for 5 min. The lysate was removed from the cell disrupter and washed with TK10ED + 0.1 M sucrose. Lysate + wash fluid were centrifuged at 4300 g for 20 min. The supernatant was set aside and the pellet was resuspended in TK10ED + 0.1 M sucrose, and subsequently centrifuged at 4300 g for 20 min. This process was repeated once more with the remaining pellet, and the three supernatants were pooled (Fraction I). Five percent polymin P, pH 7.4, was added slowly to Fraction I to a final concentration of 0.7%. The suspension was stirred for 30 min, centrifuged at 8500 g for 30 min and the pellet was stored at –80°C (Fraction II). Solid ammonium sulfate was added to Fraction II supernatant to 50% saturation (0°C) and stirred for 60 min. The precipitate was removed by centrifugation (for 30 min at 8500 g). Additional solid ammonium sulfate was added to the supernatant to 80% saturation, stirred for 60 min and the 80% precipitate was collected by centrifugation (for 30 min at 45 000 g). The 80% pellet was dissolved in PNa50ED buffer (50 mM NaH2PO4, pH 7.8, 1 mM EDTA and 1 mM DTT), dialyzed against the same buffer and loaded at 15 ml h–1 onto a CM-C50 Trisacryl (IBF) column (1.6 x 4 cm) equilibrated with PNa50ED buffer and eluted successively with PNa50ED + 100 mM NaCl, PNa50ED + 200 mM NaCl and PNa50ED + 1 M NaCl. The active fractions eluted at 200 mM NaCl. After being dialyzed against PNa10ED buffer, pH 7.2, the active fractions were loaded onto a CHT-HTP (Bio-Rad) cartridge (5 ml) equilibrated with the same buffer and eluted with a NaH2PO4 (plus EDTA and DTT) gradient from 100 mM, pH 7.2 to 400 mM, pH 6.8. The active fractions eluted at 240 mM NaH2PO4. After being dialyzed against PNa10ED buffer, pH 6.8 plus NaCl 240 mM, the active fractions were loaded onto a Heparin (Bio-Rad) cartridge (5 ml) equilibrated with the same buffer and eluted with a 240–600 mM NaCl gradient in PNa10ED buffer, pH 6.8. The active fractions eluted around 370 mM NaCl. The enzyme purified (98%) from C.guilliermondii presented a molecular mass of 90 kDa.
Other products
Negatively supercoiled pBR 322 DNA was prepared as described previously (44) Human topoisomerase I was purchased from TopoGen Inc. PLP, PL, pyridoxamine (PM), pyridoxamine-5'-phosphate (PMP) and camptothecin were from Sigma. The preparation of PLP-AMP was performed as described previously (45). The chemical structures of the PL-related compounds are shown in Figure 1.
Figure 1. Chemical structures of vitamin B6 compounds: compound 1, PL; compound 2, PLP; compound 3, PM; compound 4, PMP. Chemical structures of PLP-AMP derivative: compound 5, PLP-AMP. Chemical structures of Lys–PLP complex: compound 6, complex Lys–PLP.
Protein determination
The protein concentration was determined using Schaffner and Weissmann's method (46) with bovine serum albumin as the standard. Protein samples were analyzed on a 5–20% polyacrylamide gradient slab gel in the presence of SDS as described by Laemmli (47) and silver stained by the simplified ultra sensitive technique of Oakley et al. (48).
Enzyme assays
Topoisomerase I activity
The optimal reaction mixture was used (25). Incubation buffer, 10 mM Tris–HCl, pH 8.0, 0.5 mM DTT, 2% glycerol, contained 50 ng of negatively supercoiled DNA (pBR 322), 100 mM KCl, 8 mM MgCl2 and topoisomerase I (C.guilliermondii or Homo sapiens). The mixture was incubated at initial velocity for 2.5 min at 37°C. The enzymatic reaction was then stopped by the addition of 1/10 vol 4% lithium dodecyl sulfate, 0.3% bromophenol blue and 50% sucrose. The DNA products of relaxation were separated by horizontal agarose gel electrophoresis (49). The electrophoretic analysis was performed in 1.2% agarose slab gels (11.5 x 13.5 x 0.5 cm) in an electrophoresis buffer containing 36 mM Tris base/1 mM EDTA/30 mM sodium phosphate. Current was applied at 50 V for 4 h for relaxation with recirculation of buffer between reservoirs. Following electrophoresis, gels were stained for 15 min in ethidium bromide at 2 μg ml–1 in bidistilled water, and subsequently transferred into bidistilled water for another 15 min. Gels were then illuminated from below with a short-wave ultraviolet (UV) light plate and photographs were taken with a digital camera. Densitometric scanning of image capture enabled the activity to be quantified with Scan Analysis software as the kinetic of disappearance (%) of supercoiled DNA substrate pBR 322.
DNA cleavage assay
DNA cleavage reactions were carried out in the same incubation buffer as above, but with 10 ng of negatively supercoiled DNA (pBR 322) and 5 U of human topoisomerase I. The reactions were incubated for 15 min at 37°C, then mixed with 1% SDS and 50 μg ml–1 proteinase K and finally incubated at 50°C for 30 min. After adding 0.3% bromophenol blue and 50% sucrose, the DNA products were analyzed with the same electrophoretic system as above, except the electrophoretic buffer (Tris 100 mM, Borate 100 mM, EDTA 2 mM and 0.5 μg ml–1 of ethidium bromide) that allowed a direct visualization on the UV light plate.
Enzyme reduction by sodium borohydride
The purified C.guilliermondii topoisomerase I was incubated in the dark with PLP for 30 min at room temperature (–20°C). Stock NaBH4 (Sigma), in 0.24% NaOH solution was freshly prepared just before use. Reduction of the PLP-modified C.guilliermondii topoisomerase I was performed at 0°C in the dark for 30 min with (3H) NaBH4 in Tris–HCl buffer, pH 8.0. The final concentration of enzyme was 10–8 M, PLP was 1.8 x 10–4 M, and (3H) NaBH4 was 3.63 x 10–3 M and 15 mCi. The tritiated sample was loaded onto a Whatman GF/C glass fiber filter (2.5 cm diameter), then washed with twenty 5 ml aliquots of cold 20% trichloracetic acid prior to counting in Kontron Betamatic IV.
Spectral changes upon reduction of C.guilliermondii topoisomerase I by NaBH4
The UV/visible spectrum of 40 μg purified C.guilliermondii topoisomerase I in 50 mM Tris–HCl buffer, pH 8.0 was obtained in the presence of PLP (10-fold excess). After the addition of NaBH4 (20 mM), the mixture was incubated at 22°C for 6 min, before the spectrum of the reduced enzyme was recorded.
Tryptic digestion of the PLP-modified (3H) NaBH4-reduced C.guilliermondii topoisomerase I
Modified (3H) NaBH4-reduced C.guilliermondii topoisomerase I was prepared on a larger scale by incubating the topoisomerase I with PLP. The Schiff base complex was reduced for 30 min with freshly solubilized 5 mM (3H) NaBH4 (0.24% NaOH) at 0°C in the dark. To the washed PLP-modified (3H)-labeled topoisomerase I from C.guilliermondii, 50 μg of trypsin in 0.1 M NH4HCO3 was added, and incubation was allowed to proceed for 10 h at 37°C. The digestion mixture was lowered to pH 4 with trifluoroacetic acid to stop the proteolysis reaction. Undissolved materials were removed by centrifugation. Peptides from the trypsin proteolysis were then dried down with a Speed Vac in a vacuum. The dried peptides were redissolved in 0.1% trifluoroacetic acid.
Separation of tryptic peptides
Tryptic digests in 0.1% trifluoroacetic acid were directly loaded on a Waters (YMC-Pack ODS-AMQ, S-5 μm; 4.6 x 250 mm) C18 reverse-phase high-performance liquid chromatography (HPLC) column and eluted with 2–45% (v/v) acetonitrile gradient containing 0.1% trifluoroacetic acid at a flow rate of 200 μl min–1. Eluate absorbance was monitored at 214 nm. The most radioactive fractions emerged around 25 min containing more than 56% of the total radioactivity associated with C.guilliermondii topoisomerase I. The fractions showing radioactivity were pooled, lyophilized and subjected to the same gradient onto the same C18 reverse-phase HPLC column. Three radioactive fractions appeared where the most radioactive fractions (20 min) accounted for 74% of the total eluted radioactivity.
Amino acid sequence analysis of tryptic peptide
The most radioactive fractions were lyophilized, redissolved in 60 μl of 50% ethanol/2% acetic acid (v/v) and submitted to an Applied Biosystem Procise sequencer for an automated Edman degradation.
Molecular modeling
A molecular model of human topoisomerase I bearing the K532–PLP moiety was obtained with the molecular modeling package Insight II (Accelrys/Molecular Simulations Inc., San Diego, CA) (cff91 force field) on a Silicon Graphics, Inc., Octane R10000 computer (http://www.accelrys.com). Operations were performed starting from the conformation adopted by human topoisomerase I non-covalently complexed to a DNA duplex (PDB code 1A35 , 2.50 ? resolution) (18). In a first step, the DNA and the surrounding water molecules were removed from the complex, and the Y723F catalytic mutation introduced by authors to avoid the DNA cutting was reverted. Then, the initial K532–PLP human topoisomerase I was built in using the conformation adopted by the K39–PLP moiety in the PLP-bound alanine racemase K39–PLP (50).
To eliminate unfavorable steric interactions generated by the insertion of PLP in the enzyme active site, the resulting structure was submitted to a minimization protocol, including a step of steepest descent minimization performed until a final convergence of 2 kcal mol–1 ?–1 was reached, and a step of conjugate gradient minimization performed until a final convergence of 0.1 kcal mol–1 ?–1 was reached (Polak–Ribiere method). The non-bond part of the energy calculation used the cell multipole method, and the Coulombic interactions were calculated with a distance-dependent dielectric for which the constant was set to 1.
RESULTS
PLP as a catalytic inhibitor of DNA topoisomerase IB
The chemical structures of the PL-related compounds are illustrated in Figure 1. Inhibition of C.guilliermondii and human topoisomerases I by PLP was first tested in a DNA relaxation assay (Figure 2A). Human topoisomerase I (2 U) was still inactive in the presence of an excess of PLP after 20 min of incubation time (lane 14). Inhibition of the C.guilliermondii enzyme by PLP was quantified using an alternative method to the IC 50 determination. The global DNA change from supercoiled to relaxed topoisomers was found proportional to the incubation time, describing initial velocity conditions (25). Actually, an analysis by the Dixon method revealed that the inhibition depends on the DNA concentration in a competitive fashion (Figure 2B). A Ki value of 40 μM was directly deduced at the intercept of the plots. The inhibition specificity was assessed by the comparison of PLP with its chemically related compounds PL and PLP-AMP. Apparent Ki values of 92, 53 and 23 μM were found for PL, PLP and PLP-AMP, respectively (Figure 2C), while PM, PMP and the phosphate group alone did not display any inhibitory effect (data not shown). We deduced that the aldehyde group is required for an efficient enzyme inactivation, while the addition of a phosphate group or a phosphate AMP group slightly potentiates the effect. Moreover, the competition observed between PLP and DNA demonstrates that the binding of PLP to topoisomerase I directly affects the overall binding of the enzyme to DNA.
Figure 2. Inhibition of DNA relaxation by PL-related compounds. (A) Electrophoretic pattern of pBR322 DNA (50 ng) after inhibition of topoisomerase I relaxation activity. C.guilliermondii topoisomerase I (2 U): lanes 1–12, PLP concentrations from 0, 10, 12.5, 15, 20, 25, 30, 40, 50, 55, 60 and 75 μM for an incubation time of 2.5 min, and lane 13, 100 μM for an incubation time of 20 min. Human topoisomerase I (2 U): lane 14, PLP concentration of 140 μM for an incubation time of 20 min, and lane 15 positive control. (B and C) Dixon representation 1/v versus , and in the presence of a fixed concentration of substrate (DNA) where v (velocity) is calculated as (relaxed DNA/t) for an incubation time of 2.5 min. (B) Kinetic analysis of the inhibitory effect of PLP on C.guilliermondii topoisomerase I and determination of the Ki value with four substrate concentrations of DNA: 30 ng (open squares), 50 ng (filled circles), 80 ng (open diamonds) and 160 ng (filled triangles). (C) Kinetic analysis of the inhibitory effect of PLP (filled circles), PL (filled squares) and PLP-AMP (filled triangles) on C.guilliermondii topoisomerase I with 50 ng of DNA and determination of apparent Ki values. (D) Electrophoretic pattern of pBR322 DNA cleavage assay: reactions were incubated with pBR 322 DNA (10 ng) and human topoisomerase I (5 U) at 37°C for 15 min, then incubated with SDS (1%) and proteinase K (50 μg ml–1) at 50°C for 30 min. PLP (200 μM) and camptothecin 100 μM were added in the reaction as shown. Relaxed DNA products (Ir) were positively supercoiled in the ethidium bromide-containing gel and migrate faster than cleaved DNA (form II) and native DNA (Is). In all these assays (A–D), topoisomerase I and PL-related compounds have been preincubated for 5 min at 4°C to allow the formation of the topoisomerase I–ligand complex in the reaction buffer before adding DNA.
To better delineate the competitive inhibition of topoisomerase I by PLP, the impact of the latter was tested on the formation of topoisomerase I–DNA cleavable complex (Figure 2D). Electrophoresis on BET-containing agarose gel shows that relaxed DNA was switched to a highly supercoiled form, while negative supercoiled DNA was switched to a slightly more relaxed form. Camptothecin added in excess to the DNA relaxation assay in the presence of saturating amount of human topoisomerase I stimulated the formation of form II DNA; in same conditions, topoisomerase I preincubated with PLP did not provoke any change on DNA. Camptothecin added to PLP-saturated topoisomerase I did not produce any form II DNA, while at the same time, form I DNA kept the topological constraint of the initial DNA substrate, stipulating that the PLP-treated topoisomerase I cannot anymore act on DNA. PLP is therefore a competitive catalytic inhibitor of topoisomerase IB that interferes with the binding of the enzyme to DNA and thereby with the first transesterification.
PLP targets the critical lysine residue of topoisomerase IB
Generally, the aldehyde group of PLP reacts with the -amino group of lysines yielding an aldimine bond or a Schiff base (Figure 1) that can be further reduced by NaBH4 to create a stable covalent bond (51). Here, the reduction of the aldimine bond in the PLP-treated topoisomerase I was proved by a significant UV spectral change (Figure 3A), similar to that observed in the PLP-bound aminotransferases (52,53).
Figure 3. Identification of active site lysine and its contiguous residues by PLP. (A) The UV/visible spectrum of non-reduced C.guilliermondii topoisomerase I/PLP consists in absorption bands in both the UV and visible regions (solid line). The prominent peak at 420 nm (pH 8.0) is characteristic of aldimine formation between the bound PLP and the -amino group of a lysine residue. The 420 nm absorption is bleached by the addition of NaBH4 to the solution concomitant with an absorption increase at 325 nm (dashed line). Insert: difference spectrum of the reduced and unreduced enzyme. (B) HPLC profile of modified (3H) NaBH4-reduced C.guilliermondii topoisomerase I. The radioactive peak eluted from a first C18 reverse-phase column was loaded onto a second C18 reverse-phase HPLC column, then eluted as described in Materials and Methods. Fractions were collected and an aliquot from each withdrawn for (3H)-counting. The most radioactive fractions emerged at about 25 min as marked by an arrow in panel a. The preceding most radioactive fractions were re-chromatographed in the same conditions (see Materials and Methods). Following this step, three peptides were resolved where the more important (marked by an arrow) accounted for 74% of the total eluted radioactivity (B). (C) Sequence alignment of C.guilliermondii lysine–PLP topoisomerase I with homologous regions from other eukaryotic type I topoisomerases. The sequences are labeled as follows: H.sapiens, C.guilliermondii var. membranaefaciens, C.albicans, S.cerevisiae and S.pombe. The secondary structure elements relative to the human enzyme are displayed at the top and colored as follow: helices as dark tubes, sheets as white arrows. A white box highlights C.guilliermondii PLP-bound lysine residue.
To identify the targeted lysine, the protein–PLP complex was treated with (3H) NaBH4 to make the reduced imine bond radioactive. Experiments, repeated five times, provided a PLP incorporation of 0.96–1.14 moles per mole of holoenzyme, in agreement with a 1:1 stoechiometry. The (3H) PLP–protein was then submitted to trypsinolysis and the released peptides were separated using two successive C18 reverse-phase HPLCs. One of these peptides incorporated most of the radioactivity (Figure 3B, panels a and b), proving that it is a privileged target for the binding of PLP. The purified peptide displayed the following 17 residue sequence: KPPNTVIFDFLGK*DSIR, with the radioactivity exclusively located on lysine (K*) at position 13 (Figure 3C). The sequence displayed 64.7% identity and 76.5% similarity with the sequences found in H.sapiens (54), Schizosaccharomyces pombe (55), Saccharomyces cerevisiae (56) and Candida albicans topoisomerases IB (57) (a five residue insertion was observed in the human sequence). The C.guilliermondii lysine trapped by PLP was clearly identified as equivalent to human K532, the latter occupying the same position in a totally conserved sequence of seven residues within the isolated peptide (Figure 3C). Note that the PLP-treated human topoisomerase I also failed to relax the form I DNA and to stimulate the DNA cleavable complex (Figure 2A and D).
All together, the above features confirm several previous observations identifying the active site contained lysine of topoisomerase IB as a key residue for the catalytic activity (20,58). The high similarity displayed by the residues flanking this lysine suggests that they also adopt similar folds in all the different topoisomerases IB. It can thus be assumed that the PLP-targeted lysine of C.guilliermondii topoisomerase I is likely as critical as the active site lysine of the paradigmatical human and vaccinia topoisomerases IB (20).
Modeling of the lysine–PLP moiety in the active site of human topoisomerase I
In the available PLP-enzyme crystal structures, the PLP moiety is always involved in a large network of interactions with the protein residues (50,59–61). In the crystal structure of the PLP-alanine racemase of Bacillus stearothermophilus, the K39–PLP moiety creates interactions with a frame of active site residues similar to those of topoisomerase IB. The lysine-pyridoxal 5'-phosphate (Lys–PLP) moiety in the same conformation as in the PLP-alanine racemase (this corresponds to a minimum energy structure) was inserted in the active site cavity of the crystal structure of the human topoisomerase I–DNA complex (18) cleared of its K532 residue and of its DNA ligand. Actually, the overall fold of the resulting PLP-topoisomerase structure was not modified by minimization (Figure 4B). The Ramachandran plots (data not shown) revealed a similar low number of non-allowed combinations of phi and psi angles in the experimental structure and the minimized model. The backbone root mean-square deviations (RMSDs) between experimental and minimized structures, ranging from 1.3 to 2.5 ?, illustrate the global rearrangement accompanying the accommodation of PLP in the active site. Their comparison with the heavy atom RMSDs, ranging from 1.3 to 2.6 ?, depicts the small local movements taking place in the active site side chains. When superposing back the substrate DNA on the minimized model, the PLP phosphate group takes place very close to the DNA scissile strand (Figure 4B).
Figure 4. Minimized model of human topoisomerase I bearing the K532–PLP modification. (A) Crystal structure of human topoisomerase I non-covalently complexed to a DNA duplex (PDB code 1A35 , 2.50 ? resolution). (B) Secondary structure rendering the minimized model of human topoisomerase I bearing the K532–PLP modification with the double-stranded DNA from the experimental structure superimposed. Active site residues are displayed and colored according to atoms with the carbons in gray except for the K532–PLP carbons which are colored orange. The DNA scissile strand is colored in blue and the intact strand is colored in pink.
Trapping the general acid catalyst K532 by PL-related compounds requires a proper accommodation within the enzyme active site. Obviously, the active site, which is certainly not preformed in the free enzyme, could adopt various geometries to accommodate the DNA substrate or the different PL-related compounds. Nevertheless, our molecular modeling shows that the geometry adopted by the DNA-bound active site is very convenient for an efficient binding of PLP and therefore for trapping the K532 residue (Figures 4 and 5).
Figure 5. Stereo view of the K532–PLP minimized structure. The active site residues R488, R590, H632, Y723 and K587 are also represented. Residues are colored by atoms, with backbone carbon atoms in pink and side chain carbon atoms in orange (K532–PLP) or gray (all others). Some hydrogen atoms were not shown for clarity reasons.
DISCUSSION
Treatment of enzyme with PLP provides an attractive approach to detect critical lysines and collect fruitful information on active sites. Data presented above allowed us to specify how the binding of PLP and of its analogs to the active site lysine of a eukaryotic topoisomerase IB severely impairs its activity. Clearly, PLP promotes the enzyme inhibition not from a binding to DNA but rather from a direct interaction with a lysine residue required for the formation of the cleavage complex. No specificity for a given lysine, similar to that found in C.guilliermondii topoisomerase I, has been observed in the various DNA polymerases so far tested (34,37,38). This could be explained by the low pK of the lysine -amino group in the acidic microenvironment of the active site. Remember that this lysine residue displays general acid catalyst properties essential to the formation of the topoisomerase IB–DNA cleavable complex (19,20). In forming an aldimine with lysine, PLP inhibits the DNA cleavage in impeding the binding of the enzyme to DNA and then the first transesterification step. Such properties bring to light PLP and its derivatives as a new class of topoisomerase IB catalytic inhibitors.
Here, the Dixon plot method was used for the first time to evaluate the competitive inhibition of DNA relaxation by DNA topoisomerases (a value of 40 μM was obtained for PLP). Preincubation of the enzyme with the PL-related derivatives prior to the addition of the pBR322 DNA was required to observe inhibition. The latter becomes clearly weaker when PLP and DNA are added together to the reaction mixture (data not shown), suggesting that the binding of PLP to the free enzyme is slower than the formation of a productive enzyme–DNA complex, or/and that the DNA-bound enzyme is less accessible. Actually, two non-exclusive hypotheses might explain the competition between PLP and DNA: (i) the PLP-bound enzyme only poorly binds to DNA and (ii) the binding of DNA to the PLP–enzyme complex induces the PLP dissociation. To understand how PLP and its derivatives bind to topoisomerase IB and compete with DNA now requires the use of a single turnover assay with a small DNA substrate that will provide the kinetic parameters.
Another point is the reversibility of the Schiff base reaction. Competition between DNA and PLP for binding to topoisomerase IB requires that the PLP binding to topoisomerase IB is reversible under non-reducing conditions. As shown in the case of several dehydrogenases (62,63), the internal imine bond between PLP and the -amino group of a lysine residue is in equilibrium between a non-covalent and a covalent form. The formation of an internal aldimine generally depends on pH and solvent and can be dissociated by dialysis, dilution or by adding hydroxylamine to the incubation buffer (64). Here, the addition of DNA to a sample of PLP preincubated with the enzyme increased the ratio of active enzyme, proving that DNA can dissociate PLP from its binding site.
Topoisomerase IB displays a typical range of inhibition effects for PLP and PLP-AMP. The small differences between the apparent Ki of PLP (52 μM) and of PLP-AMP (23 μM) could be explained by different binding patterns. A noticeable point is that PL is significantly active against topoisomerase IB (Ki of 92 μM), when it is much less active against several other proteins, including DNA polymerases (65). Different conformational fits may allow the sequestration of PL, PLP and PLP-AMP with an increasing number of interactions.
Obviously, the PLP-topoisomerase IB model needs to be ascertained either by a full molecular dynamics calculation taking into account solvation or by crystal structure analysis of a modified human topoisomerase IB. Such studies coupled with single turnover and DNA-binding assays in the presence of PL-related compounds will allow a better understanding of the conformational changes accompanying the DNA recognition and the protein clamp formation events (66) that enable catalysis initiation.
According to recent data, the PLP-vitamin B6 in supra-physiological amount exerts anti-proliferative (40) and anti-tumoral (41) activities. These activities have been attributed to the inhibition of DNA topoisomerases I and II as well as of replicating DNA polymerases and (40,65). Even if the in vivo selectivity of PLP against topoisomerase IB should be questioned, it represents a possible new lead compound ought to its original mode of inhibition by direct prevention of the chemical catalysis. The use of molecular modeling that permits to address the structural modifications affecting the active site upon ligand binding (67) could help to design new PLP analogs with better affinity and selectivity for topoisomerase IB.
Finally, why does PLP and its derivatives bind the topoisomerase I active site? A speculation would be that PLP exerts a physiological negative regulation of the topoisomerase IB activity with a possible impact on its intracellular location. An analysis of the intracellular uptake and availability of PLP together with an exhaustive search of the PLP-binding proteins within the eukaryotic cells is needed to determine whether the topoisomerase IB is a natural PLP-binding enzyme.
ACKNOWLEDGEMENTS
We are indebted to Jacques d'Alayer (Institut Pasteur) for peptide analysis, Prof. Paul Cohen for the use of laboratory facilities, Drs Hugues-Olivier Bertrand, Anne Goupil-Lamy and Remy Hoffmann from Accelrys SARL and Société Alain Mikli (Paris) for the financial support. This work was supported by grants from Université Pierre et Marie Curie, CNRS, French Ministry of Research and Accelrys SARL (S.C.F.).
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* To whom correspondence should be addressed. Tel: +331 53 63 40 76; Fax: +331 53 63 40 77; Email: dergara@ccr.jussieu.fr
ABSTRACT
The present results demonstrate that pyridoxal, pyridoxal 5'-phosphate (PLP) and pyridoxal 5'-diphospho-5'-adenosine (PLP-AMP) inhibit Candida guilliermondii and human DNA topoisomerases I in forming an aldimine with the -amino group of an active site lysine. PLP acts as a competitive inhibitor of C.guilliermondii topoisomerase I (Ki = 40 μM) that blocks the cleavable complex formation. Chemical reduction of PLP-treated enzyme reveals incorporation of 1 mol of PLP per mol of protein. The limited trypsic proteolysis releases a 17 residue peptide bearing a lysine-bound PLP (KPPNTVIFDFLGK*DSIR). Targeted lysine (K*) in C.guilliermondii topoisomerase I corresponds to that found in topoisomerase I of Homo sapiens (K532), Candida albicans (K468), Saccharomyces cerevisiae (K458) and Schizosaccharomyces pombe (K505). In the human enzyme, K532, belonging to the active site acts as a general acid catalyst and is therefore essential for activity. The spatial orientation of K532–PLP within the active site was approached by molecular modeling using available crystallographic data. The PLP moiety was found at close proximity of several active residues. PLP could be involved in the cellular control of topoisomerases IB. It constitutes an efficient tool to explore topoisomerase IB dynamics during catalysis and is also a lead for new drugs that trap the lysine general acid.
INTRODUCTION
The DNA topoisomerases I and II are key enzymes that control the DNA topological states during DNA replication, transcription, repair, recombination and chromatin remodeling. Eukaryotic topoisomerases IA and IB catalyze topological rearrangements of DNA through cleavage and religation of one DNA strand (1–4), either by passing the unbroken strand through the break (topoisomerases IA) (5) or by rotating the broken strand around the unbroken strand (topoisomerases IB) (6,7). The sequential mechanism of the strand rotation controlled by eukaryotic topoisomerases IB involves the reversible formation of a 3'-O-phosphoro-tyrosine bond between the active site tyrosine and the cleaved DNA (8). DNA cleavage and religation can be separated using partial DNA duplexes (9–11). The anti-tumoral derivatives of camptothecin, including the water soluble topotecan used for the treatment of ovarian and colon carcinoma (12,13), stall topoisomerases IB on the DNA between the cleavage and religation steps (14) by intercalation into the active-site-bound DNA (15).
The crystal structure of human topoisomerase I non-covalently bound to DNA reveals that the bilobed enzyme completely wraps around its DNA cognate (7,16–18) with a vast majority of the contacts occurring with the phosphate backbone. Only the base-specific interaction concerns the -amino group of the conserved K532 and the base O2 atom of the consensual thymine on the scissile strand, upstream from the cleavage site (position –1). This interaction has been proposed to stabilize a transition state required for the strand alignment during the religation step (19). The conserved catalytic Y723 and the positively charged residues R488, R590 and H632 occupy positions near the scissile phosphate group so that Y723 can attack the scissile phosphate group, while R488, R590 and H632 stabilize the transition state of the scissile phosphate, and K532 (equivalent to vaccinia K167) acts as the general acid catalyst (20,21). The enzyme conformation that results from binding to DNA allows Y723 to join the active site (22,23). The DNA controlled rotation of topoisomerase IB requires specific concerted motions (24), but these are hindered by intercalation of topotecan into the active site-bound DNA (15). Its global processivity is greatly enhanced in vitro by weak anions and magnesium (25). A concerted mechanism of proton relay has been proven essential in vaccinia topoisomerase I during the transesterification steps that control the DNA strand break, involving the residues K167, R130 (the latter being equivalent to human R488) and the DNA 5' oxygen of the scissile phosphate (26). The crucial role of a lysine residue side chain in this proton relay has been recently confirmed with the K532 residue of human topoisomerase IB (19). Protonation of the DNA 5' oxygen facilitates the nucleophilic attack of the scissile phosphate by Y723.
Topoisomerase IB is a recognized target for anti-cancer drugs (27,28). Camptothecin represents the prototype of a large class of genotoxic drugs that act as topoisomerase IB poisons, while a second class, acting as topoisomerase I suppressors, consists in catalytic inhibitors that compete with DNA to bind to the enzyme. For such drugs, the relationships between the enzyme inactivation and the anti-proliferative effects are still intriguing as in the presence of a topoisomerase I poison they usually prevent the cleavable complex formation whereas, alone, they exhibit genotoxic properties in cultured cells compound (29)].
The vitamin B6-related pyridoxal 5'-phosphate (PLP) is a lysine-binding coenzyme for a number of enzymes involved in amino acid metabolism, including aminotransferases (30). It also binds to enzymes that recognize phosphate-containing substrates or effectors, allowing its use to probe critical lysine residues within the dNTP-binding domain of DNA polymerases (31–34), RNA polymerases (35–37) and HIV reverse transcriptase (38). We have previously reported that PLP acts as an efficient inhibitor of the C.guilliermondii topoisomerase I (39). Using PLP, pyridoxal (PL) and pyridoxal 5'-diphospho-5'-adenosine (PLP-AMP), we now confirm the critical catalytic function of a lysine residue in C.guilliermondii topoisomerase I, equivalent to residue K532 of the human topoisomerase IB active site (20,21). We demonstrate that PLP is a catalytic inhibitor that competes with the binding of the enzyme to DNA in forming a Schiff base with a lysine residue of the active site. PLP that has been already shown in supra-physiological amount to possess anti-proliferative (40) and anti-tumoral (41) activities could therefore belong to the class of topoisomerase IB inhibitors. Molecular modeling based on the active site of the human topoisomerase I crystal structure (42) provides some information on the spatial organization of PLP adducted on the critical lysine.
MATERIALS AND METHODS
Purification of topoisomerase I from C.guilliermondii (25)
Unless otherwise specified, the purification was carried out at 4°C. C.guilliermondii var. membranaefaciens was cultured as described previously (43). The C.guilliermondii cells were suspended in a buffer containing 50 mM Tris–HCl, pH 7.4, 10 mM KCl, 1 mM EDTA, 1 mM DTT (TK10ED) plus 0.1 M sucrose and lysed in a Dyno Mill disrupter (Willy A. Bachofen AG Maschinenfabrik, Basel, Switzerland) at 3000 r.p.m. for 5 min. The lysate was removed from the cell disrupter and washed with TK10ED + 0.1 M sucrose. Lysate + wash fluid were centrifuged at 4300 g for 20 min. The supernatant was set aside and the pellet was resuspended in TK10ED + 0.1 M sucrose, and subsequently centrifuged at 4300 g for 20 min. This process was repeated once more with the remaining pellet, and the three supernatants were pooled (Fraction I). Five percent polymin P, pH 7.4, was added slowly to Fraction I to a final concentration of 0.7%. The suspension was stirred for 30 min, centrifuged at 8500 g for 30 min and the pellet was stored at –80°C (Fraction II). Solid ammonium sulfate was added to Fraction II supernatant to 50% saturation (0°C) and stirred for 60 min. The precipitate was removed by centrifugation (for 30 min at 8500 g). Additional solid ammonium sulfate was added to the supernatant to 80% saturation, stirred for 60 min and the 80% precipitate was collected by centrifugation (for 30 min at 45 000 g). The 80% pellet was dissolved in PNa50ED buffer (50 mM NaH2PO4, pH 7.8, 1 mM EDTA and 1 mM DTT), dialyzed against the same buffer and loaded at 15 ml h–1 onto a CM-C50 Trisacryl (IBF) column (1.6 x 4 cm) equilibrated with PNa50ED buffer and eluted successively with PNa50ED + 100 mM NaCl, PNa50ED + 200 mM NaCl and PNa50ED + 1 M NaCl. The active fractions eluted at 200 mM NaCl. After being dialyzed against PNa10ED buffer, pH 7.2, the active fractions were loaded onto a CHT-HTP (Bio-Rad) cartridge (5 ml) equilibrated with the same buffer and eluted with a NaH2PO4 (plus EDTA and DTT) gradient from 100 mM, pH 7.2 to 400 mM, pH 6.8. The active fractions eluted at 240 mM NaH2PO4. After being dialyzed against PNa10ED buffer, pH 6.8 plus NaCl 240 mM, the active fractions were loaded onto a Heparin (Bio-Rad) cartridge (5 ml) equilibrated with the same buffer and eluted with a 240–600 mM NaCl gradient in PNa10ED buffer, pH 6.8. The active fractions eluted around 370 mM NaCl. The enzyme purified (98%) from C.guilliermondii presented a molecular mass of 90 kDa.
Other products
Negatively supercoiled pBR 322 DNA was prepared as described previously (44) Human topoisomerase I was purchased from TopoGen Inc. PLP, PL, pyridoxamine (PM), pyridoxamine-5'-phosphate (PMP) and camptothecin were from Sigma. The preparation of PLP-AMP was performed as described previously (45). The chemical structures of the PL-related compounds are shown in Figure 1.
Figure 1. Chemical structures of vitamin B6 compounds: compound 1, PL; compound 2, PLP; compound 3, PM; compound 4, PMP. Chemical structures of PLP-AMP derivative: compound 5, PLP-AMP. Chemical structures of Lys–PLP complex: compound 6, complex Lys–PLP.
Protein determination
The protein concentration was determined using Schaffner and Weissmann's method (46) with bovine serum albumin as the standard. Protein samples were analyzed on a 5–20% polyacrylamide gradient slab gel in the presence of SDS as described by Laemmli (47) and silver stained by the simplified ultra sensitive technique of Oakley et al. (48).
Enzyme assays
Topoisomerase I activity
The optimal reaction mixture was used (25). Incubation buffer, 10 mM Tris–HCl, pH 8.0, 0.5 mM DTT, 2% glycerol, contained 50 ng of negatively supercoiled DNA (pBR 322), 100 mM KCl, 8 mM MgCl2 and topoisomerase I (C.guilliermondii or Homo sapiens). The mixture was incubated at initial velocity for 2.5 min at 37°C. The enzymatic reaction was then stopped by the addition of 1/10 vol 4% lithium dodecyl sulfate, 0.3% bromophenol blue and 50% sucrose. The DNA products of relaxation were separated by horizontal agarose gel electrophoresis (49). The electrophoretic analysis was performed in 1.2% agarose slab gels (11.5 x 13.5 x 0.5 cm) in an electrophoresis buffer containing 36 mM Tris base/1 mM EDTA/30 mM sodium phosphate. Current was applied at 50 V for 4 h for relaxation with recirculation of buffer between reservoirs. Following electrophoresis, gels were stained for 15 min in ethidium bromide at 2 μg ml–1 in bidistilled water, and subsequently transferred into bidistilled water for another 15 min. Gels were then illuminated from below with a short-wave ultraviolet (UV) light plate and photographs were taken with a digital camera. Densitometric scanning of image capture enabled the activity to be quantified with Scan Analysis software as the kinetic of disappearance (%) of supercoiled DNA substrate pBR 322.
DNA cleavage assay
DNA cleavage reactions were carried out in the same incubation buffer as above, but with 10 ng of negatively supercoiled DNA (pBR 322) and 5 U of human topoisomerase I. The reactions were incubated for 15 min at 37°C, then mixed with 1% SDS and 50 μg ml–1 proteinase K and finally incubated at 50°C for 30 min. After adding 0.3% bromophenol blue and 50% sucrose, the DNA products were analyzed with the same electrophoretic system as above, except the electrophoretic buffer (Tris 100 mM, Borate 100 mM, EDTA 2 mM and 0.5 μg ml–1 of ethidium bromide) that allowed a direct visualization on the UV light plate.
Enzyme reduction by sodium borohydride
The purified C.guilliermondii topoisomerase I was incubated in the dark with PLP for 30 min at room temperature (–20°C). Stock NaBH4 (Sigma), in 0.24% NaOH solution was freshly prepared just before use. Reduction of the PLP-modified C.guilliermondii topoisomerase I was performed at 0°C in the dark for 30 min with (3H) NaBH4 in Tris–HCl buffer, pH 8.0. The final concentration of enzyme was 10–8 M, PLP was 1.8 x 10–4 M, and (3H) NaBH4 was 3.63 x 10–3 M and 15 mCi. The tritiated sample was loaded onto a Whatman GF/C glass fiber filter (2.5 cm diameter), then washed with twenty 5 ml aliquots of cold 20% trichloracetic acid prior to counting in Kontron Betamatic IV.
Spectral changes upon reduction of C.guilliermondii topoisomerase I by NaBH4
The UV/visible spectrum of 40 μg purified C.guilliermondii topoisomerase I in 50 mM Tris–HCl buffer, pH 8.0 was obtained in the presence of PLP (10-fold excess). After the addition of NaBH4 (20 mM), the mixture was incubated at 22°C for 6 min, before the spectrum of the reduced enzyme was recorded.
Tryptic digestion of the PLP-modified (3H) NaBH4-reduced C.guilliermondii topoisomerase I
Modified (3H) NaBH4-reduced C.guilliermondii topoisomerase I was prepared on a larger scale by incubating the topoisomerase I with PLP. The Schiff base complex was reduced for 30 min with freshly solubilized 5 mM (3H) NaBH4 (0.24% NaOH) at 0°C in the dark. To the washed PLP-modified (3H)-labeled topoisomerase I from C.guilliermondii, 50 μg of trypsin in 0.1 M NH4HCO3 was added, and incubation was allowed to proceed for 10 h at 37°C. The digestion mixture was lowered to pH 4 with trifluoroacetic acid to stop the proteolysis reaction. Undissolved materials were removed by centrifugation. Peptides from the trypsin proteolysis were then dried down with a Speed Vac in a vacuum. The dried peptides were redissolved in 0.1% trifluoroacetic acid.
Separation of tryptic peptides
Tryptic digests in 0.1% trifluoroacetic acid were directly loaded on a Waters (YMC-Pack ODS-AMQ, S-5 μm; 4.6 x 250 mm) C18 reverse-phase high-performance liquid chromatography (HPLC) column and eluted with 2–45% (v/v) acetonitrile gradient containing 0.1% trifluoroacetic acid at a flow rate of 200 μl min–1. Eluate absorbance was monitored at 214 nm. The most radioactive fractions emerged around 25 min containing more than 56% of the total radioactivity associated with C.guilliermondii topoisomerase I. The fractions showing radioactivity were pooled, lyophilized and subjected to the same gradient onto the same C18 reverse-phase HPLC column. Three radioactive fractions appeared where the most radioactive fractions (20 min) accounted for 74% of the total eluted radioactivity.
Amino acid sequence analysis of tryptic peptide
The most radioactive fractions were lyophilized, redissolved in 60 μl of 50% ethanol/2% acetic acid (v/v) and submitted to an Applied Biosystem Procise sequencer for an automated Edman degradation.
Molecular modeling
A molecular model of human topoisomerase I bearing the K532–PLP moiety was obtained with the molecular modeling package Insight II (Accelrys/Molecular Simulations Inc., San Diego, CA) (cff91 force field) on a Silicon Graphics, Inc., Octane R10000 computer (http://www.accelrys.com). Operations were performed starting from the conformation adopted by human topoisomerase I non-covalently complexed to a DNA duplex (PDB code 1A35 , 2.50 ? resolution) (18). In a first step, the DNA and the surrounding water molecules were removed from the complex, and the Y723F catalytic mutation introduced by authors to avoid the DNA cutting was reverted. Then, the initial K532–PLP human topoisomerase I was built in using the conformation adopted by the K39–PLP moiety in the PLP-bound alanine racemase K39–PLP (50).
To eliminate unfavorable steric interactions generated by the insertion of PLP in the enzyme active site, the resulting structure was submitted to a minimization protocol, including a step of steepest descent minimization performed until a final convergence of 2 kcal mol–1 ?–1 was reached, and a step of conjugate gradient minimization performed until a final convergence of 0.1 kcal mol–1 ?–1 was reached (Polak–Ribiere method). The non-bond part of the energy calculation used the cell multipole method, and the Coulombic interactions were calculated with a distance-dependent dielectric for which the constant was set to 1.
RESULTS
PLP as a catalytic inhibitor of DNA topoisomerase IB
The chemical structures of the PL-related compounds are illustrated in Figure 1. Inhibition of C.guilliermondii and human topoisomerases I by PLP was first tested in a DNA relaxation assay (Figure 2A). Human topoisomerase I (2 U) was still inactive in the presence of an excess of PLP after 20 min of incubation time (lane 14). Inhibition of the C.guilliermondii enzyme by PLP was quantified using an alternative method to the IC 50 determination. The global DNA change from supercoiled to relaxed topoisomers was found proportional to the incubation time, describing initial velocity conditions (25). Actually, an analysis by the Dixon method revealed that the inhibition depends on the DNA concentration in a competitive fashion (Figure 2B). A Ki value of 40 μM was directly deduced at the intercept of the plots. The inhibition specificity was assessed by the comparison of PLP with its chemically related compounds PL and PLP-AMP. Apparent Ki values of 92, 53 and 23 μM were found for PL, PLP and PLP-AMP, respectively (Figure 2C), while PM, PMP and the phosphate group alone did not display any inhibitory effect (data not shown). We deduced that the aldehyde group is required for an efficient enzyme inactivation, while the addition of a phosphate group or a phosphate AMP group slightly potentiates the effect. Moreover, the competition observed between PLP and DNA demonstrates that the binding of PLP to topoisomerase I directly affects the overall binding of the enzyme to DNA.
Figure 2. Inhibition of DNA relaxation by PL-related compounds. (A) Electrophoretic pattern of pBR322 DNA (50 ng) after inhibition of topoisomerase I relaxation activity. C.guilliermondii topoisomerase I (2 U): lanes 1–12, PLP concentrations from 0, 10, 12.5, 15, 20, 25, 30, 40, 50, 55, 60 and 75 μM for an incubation time of 2.5 min, and lane 13, 100 μM for an incubation time of 20 min. Human topoisomerase I (2 U): lane 14, PLP concentration of 140 μM for an incubation time of 20 min, and lane 15 positive control. (B and C) Dixon representation 1/v versus , and in the presence of a fixed concentration of substrate (DNA) where v (velocity) is calculated as (relaxed DNA/t) for an incubation time of 2.5 min. (B) Kinetic analysis of the inhibitory effect of PLP on C.guilliermondii topoisomerase I and determination of the Ki value with four substrate concentrations of DNA: 30 ng (open squares), 50 ng (filled circles), 80 ng (open diamonds) and 160 ng (filled triangles). (C) Kinetic analysis of the inhibitory effect of PLP (filled circles), PL (filled squares) and PLP-AMP (filled triangles) on C.guilliermondii topoisomerase I with 50 ng of DNA and determination of apparent Ki values. (D) Electrophoretic pattern of pBR322 DNA cleavage assay: reactions were incubated with pBR 322 DNA (10 ng) and human topoisomerase I (5 U) at 37°C for 15 min, then incubated with SDS (1%) and proteinase K (50 μg ml–1) at 50°C for 30 min. PLP (200 μM) and camptothecin 100 μM were added in the reaction as shown. Relaxed DNA products (Ir) were positively supercoiled in the ethidium bromide-containing gel and migrate faster than cleaved DNA (form II) and native DNA (Is). In all these assays (A–D), topoisomerase I and PL-related compounds have been preincubated for 5 min at 4°C to allow the formation of the topoisomerase I–ligand complex in the reaction buffer before adding DNA.
To better delineate the competitive inhibition of topoisomerase I by PLP, the impact of the latter was tested on the formation of topoisomerase I–DNA cleavable complex (Figure 2D). Electrophoresis on BET-containing agarose gel shows that relaxed DNA was switched to a highly supercoiled form, while negative supercoiled DNA was switched to a slightly more relaxed form. Camptothecin added in excess to the DNA relaxation assay in the presence of saturating amount of human topoisomerase I stimulated the formation of form II DNA; in same conditions, topoisomerase I preincubated with PLP did not provoke any change on DNA. Camptothecin added to PLP-saturated topoisomerase I did not produce any form II DNA, while at the same time, form I DNA kept the topological constraint of the initial DNA substrate, stipulating that the PLP-treated topoisomerase I cannot anymore act on DNA. PLP is therefore a competitive catalytic inhibitor of topoisomerase IB that interferes with the binding of the enzyme to DNA and thereby with the first transesterification.
PLP targets the critical lysine residue of topoisomerase IB
Generally, the aldehyde group of PLP reacts with the -amino group of lysines yielding an aldimine bond or a Schiff base (Figure 1) that can be further reduced by NaBH4 to create a stable covalent bond (51). Here, the reduction of the aldimine bond in the PLP-treated topoisomerase I was proved by a significant UV spectral change (Figure 3A), similar to that observed in the PLP-bound aminotransferases (52,53).
Figure 3. Identification of active site lysine and its contiguous residues by PLP. (A) The UV/visible spectrum of non-reduced C.guilliermondii topoisomerase I/PLP consists in absorption bands in both the UV and visible regions (solid line). The prominent peak at 420 nm (pH 8.0) is characteristic of aldimine formation between the bound PLP and the -amino group of a lysine residue. The 420 nm absorption is bleached by the addition of NaBH4 to the solution concomitant with an absorption increase at 325 nm (dashed line). Insert: difference spectrum of the reduced and unreduced enzyme. (B) HPLC profile of modified (3H) NaBH4-reduced C.guilliermondii topoisomerase I. The radioactive peak eluted from a first C18 reverse-phase column was loaded onto a second C18 reverse-phase HPLC column, then eluted as described in Materials and Methods. Fractions were collected and an aliquot from each withdrawn for (3H)-counting. The most radioactive fractions emerged at about 25 min as marked by an arrow in panel a. The preceding most radioactive fractions were re-chromatographed in the same conditions (see Materials and Methods). Following this step, three peptides were resolved where the more important (marked by an arrow) accounted for 74% of the total eluted radioactivity (B). (C) Sequence alignment of C.guilliermondii lysine–PLP topoisomerase I with homologous regions from other eukaryotic type I topoisomerases. The sequences are labeled as follows: H.sapiens, C.guilliermondii var. membranaefaciens, C.albicans, S.cerevisiae and S.pombe. The secondary structure elements relative to the human enzyme are displayed at the top and colored as follow: helices as dark tubes, sheets as white arrows. A white box highlights C.guilliermondii PLP-bound lysine residue.
To identify the targeted lysine, the protein–PLP complex was treated with (3H) NaBH4 to make the reduced imine bond radioactive. Experiments, repeated five times, provided a PLP incorporation of 0.96–1.14 moles per mole of holoenzyme, in agreement with a 1:1 stoechiometry. The (3H) PLP–protein was then submitted to trypsinolysis and the released peptides were separated using two successive C18 reverse-phase HPLCs. One of these peptides incorporated most of the radioactivity (Figure 3B, panels a and b), proving that it is a privileged target for the binding of PLP. The purified peptide displayed the following 17 residue sequence: KPPNTVIFDFLGK*DSIR, with the radioactivity exclusively located on lysine (K*) at position 13 (Figure 3C). The sequence displayed 64.7% identity and 76.5% similarity with the sequences found in H.sapiens (54), Schizosaccharomyces pombe (55), Saccharomyces cerevisiae (56) and Candida albicans topoisomerases IB (57) (a five residue insertion was observed in the human sequence). The C.guilliermondii lysine trapped by PLP was clearly identified as equivalent to human K532, the latter occupying the same position in a totally conserved sequence of seven residues within the isolated peptide (Figure 3C). Note that the PLP-treated human topoisomerase I also failed to relax the form I DNA and to stimulate the DNA cleavable complex (Figure 2A and D).
All together, the above features confirm several previous observations identifying the active site contained lysine of topoisomerase IB as a key residue for the catalytic activity (20,58). The high similarity displayed by the residues flanking this lysine suggests that they also adopt similar folds in all the different topoisomerases IB. It can thus be assumed that the PLP-targeted lysine of C.guilliermondii topoisomerase I is likely as critical as the active site lysine of the paradigmatical human and vaccinia topoisomerases IB (20).
Modeling of the lysine–PLP moiety in the active site of human topoisomerase I
In the available PLP-enzyme crystal structures, the PLP moiety is always involved in a large network of interactions with the protein residues (50,59–61). In the crystal structure of the PLP-alanine racemase of Bacillus stearothermophilus, the K39–PLP moiety creates interactions with a frame of active site residues similar to those of topoisomerase IB. The lysine-pyridoxal 5'-phosphate (Lys–PLP) moiety in the same conformation as in the PLP-alanine racemase (this corresponds to a minimum energy structure) was inserted in the active site cavity of the crystal structure of the human topoisomerase I–DNA complex (18) cleared of its K532 residue and of its DNA ligand. Actually, the overall fold of the resulting PLP-topoisomerase structure was not modified by minimization (Figure 4B). The Ramachandran plots (data not shown) revealed a similar low number of non-allowed combinations of phi and psi angles in the experimental structure and the minimized model. The backbone root mean-square deviations (RMSDs) between experimental and minimized structures, ranging from 1.3 to 2.5 ?, illustrate the global rearrangement accompanying the accommodation of PLP in the active site. Their comparison with the heavy atom RMSDs, ranging from 1.3 to 2.6 ?, depicts the small local movements taking place in the active site side chains. When superposing back the substrate DNA on the minimized model, the PLP phosphate group takes place very close to the DNA scissile strand (Figure 4B).
Figure 4. Minimized model of human topoisomerase I bearing the K532–PLP modification. (A) Crystal structure of human topoisomerase I non-covalently complexed to a DNA duplex (PDB code 1A35 , 2.50 ? resolution). (B) Secondary structure rendering the minimized model of human topoisomerase I bearing the K532–PLP modification with the double-stranded DNA from the experimental structure superimposed. Active site residues are displayed and colored according to atoms with the carbons in gray except for the K532–PLP carbons which are colored orange. The DNA scissile strand is colored in blue and the intact strand is colored in pink.
Trapping the general acid catalyst K532 by PL-related compounds requires a proper accommodation within the enzyme active site. Obviously, the active site, which is certainly not preformed in the free enzyme, could adopt various geometries to accommodate the DNA substrate or the different PL-related compounds. Nevertheless, our molecular modeling shows that the geometry adopted by the DNA-bound active site is very convenient for an efficient binding of PLP and therefore for trapping the K532 residue (Figures 4 and 5).
Figure 5. Stereo view of the K532–PLP minimized structure. The active site residues R488, R590, H632, Y723 and K587 are also represented. Residues are colored by atoms, with backbone carbon atoms in pink and side chain carbon atoms in orange (K532–PLP) or gray (all others). Some hydrogen atoms were not shown for clarity reasons.
DISCUSSION
Treatment of enzyme with PLP provides an attractive approach to detect critical lysines and collect fruitful information on active sites. Data presented above allowed us to specify how the binding of PLP and of its analogs to the active site lysine of a eukaryotic topoisomerase IB severely impairs its activity. Clearly, PLP promotes the enzyme inhibition not from a binding to DNA but rather from a direct interaction with a lysine residue required for the formation of the cleavage complex. No specificity for a given lysine, similar to that found in C.guilliermondii topoisomerase I, has been observed in the various DNA polymerases so far tested (34,37,38). This could be explained by the low pK of the lysine -amino group in the acidic microenvironment of the active site. Remember that this lysine residue displays general acid catalyst properties essential to the formation of the topoisomerase IB–DNA cleavable complex (19,20). In forming an aldimine with lysine, PLP inhibits the DNA cleavage in impeding the binding of the enzyme to DNA and then the first transesterification step. Such properties bring to light PLP and its derivatives as a new class of topoisomerase IB catalytic inhibitors.
Here, the Dixon plot method was used for the first time to evaluate the competitive inhibition of DNA relaxation by DNA topoisomerases (a value of 40 μM was obtained for PLP). Preincubation of the enzyme with the PL-related derivatives prior to the addition of the pBR322 DNA was required to observe inhibition. The latter becomes clearly weaker when PLP and DNA are added together to the reaction mixture (data not shown), suggesting that the binding of PLP to the free enzyme is slower than the formation of a productive enzyme–DNA complex, or/and that the DNA-bound enzyme is less accessible. Actually, two non-exclusive hypotheses might explain the competition between PLP and DNA: (i) the PLP-bound enzyme only poorly binds to DNA and (ii) the binding of DNA to the PLP–enzyme complex induces the PLP dissociation. To understand how PLP and its derivatives bind to topoisomerase IB and compete with DNA now requires the use of a single turnover assay with a small DNA substrate that will provide the kinetic parameters.
Another point is the reversibility of the Schiff base reaction. Competition between DNA and PLP for binding to topoisomerase IB requires that the PLP binding to topoisomerase IB is reversible under non-reducing conditions. As shown in the case of several dehydrogenases (62,63), the internal imine bond between PLP and the -amino group of a lysine residue is in equilibrium between a non-covalent and a covalent form. The formation of an internal aldimine generally depends on pH and solvent and can be dissociated by dialysis, dilution or by adding hydroxylamine to the incubation buffer (64). Here, the addition of DNA to a sample of PLP preincubated with the enzyme increased the ratio of active enzyme, proving that DNA can dissociate PLP from its binding site.
Topoisomerase IB displays a typical range of inhibition effects for PLP and PLP-AMP. The small differences between the apparent Ki of PLP (52 μM) and of PLP-AMP (23 μM) could be explained by different binding patterns. A noticeable point is that PL is significantly active against topoisomerase IB (Ki of 92 μM), when it is much less active against several other proteins, including DNA polymerases (65). Different conformational fits may allow the sequestration of PL, PLP and PLP-AMP with an increasing number of interactions.
Obviously, the PLP-topoisomerase IB model needs to be ascertained either by a full molecular dynamics calculation taking into account solvation or by crystal structure analysis of a modified human topoisomerase IB. Such studies coupled with single turnover and DNA-binding assays in the presence of PL-related compounds will allow a better understanding of the conformational changes accompanying the DNA recognition and the protein clamp formation events (66) that enable catalysis initiation.
According to recent data, the PLP-vitamin B6 in supra-physiological amount exerts anti-proliferative (40) and anti-tumoral (41) activities. These activities have been attributed to the inhibition of DNA topoisomerases I and II as well as of replicating DNA polymerases and (40,65). Even if the in vivo selectivity of PLP against topoisomerase IB should be questioned, it represents a possible new lead compound ought to its original mode of inhibition by direct prevention of the chemical catalysis. The use of molecular modeling that permits to address the structural modifications affecting the active site upon ligand binding (67) could help to design new PLP analogs with better affinity and selectivity for topoisomerase IB.
Finally, why does PLP and its derivatives bind the topoisomerase I active site? A speculation would be that PLP exerts a physiological negative regulation of the topoisomerase IB activity with a possible impact on its intracellular location. An analysis of the intracellular uptake and availability of PLP together with an exhaustive search of the PLP-binding proteins within the eukaryotic cells is needed to determine whether the topoisomerase IB is a natural PLP-binding enzyme.
ACKNOWLEDGEMENTS
We are indebted to Jacques d'Alayer (Institut Pasteur) for peptide analysis, Prof. Paul Cohen for the use of laboratory facilities, Drs Hugues-Olivier Bertrand, Anne Goupil-Lamy and Remy Hoffmann from Accelrys SARL and Société Alain Mikli (Paris) for the financial support. This work was supported by grants from Université Pierre et Marie Curie, CNRS, French Ministry of Research and Accelrys SARL (S.C.F.).
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