Phytochemistry xxx (2015) xxx–xxx

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Structural insight of DNA topoisomerases I from camptothecinproducing plants revealed by molecular dynamics simulations Supaart Sirikantaramas a,b,⇑, Arthitaya Meeprasert a, Thanyada Rungrotmongkol a, Hideyoshi Fuji c, Tyuji Hoshino c, Hiroshi Sudo b,d, Mami Yamazaki b, Kazuki Saito b,⇑ a

Department of Biochemistry, Faculty of Science, Chulalongkorn University, Thailand Department of Molecular Biology and Biotechnology, Graduate School of Pharmaceutical Sciences, Chiba University, Japan Department of Physical Chemistry, Graduate School of Pharmaceutical Sciences, Chiba University, Japan d Faculty of Pharmaceutical Sciences, Hoshi University, Japan b c

a r t i c l e

i n f o

Article history: Available online xxxx Dedicated to Professor Vince de Luca on the occasion of his 60th birthday. Keywords: Nothapodytes nimmoniana Icacinaceae Topoisomerase I Camptothecin Resistance Mutation

a b s t r a c t DNA topoisomerase I (Top1) catalyzes changes in DNA topology by cleaving and rejoining one strand of the double stranded (ds)DNA. Eukaryotic Top1s are the cellular target of the plant-derived anticancer indole alkaloid camptothecin (CPT), which reversibly stabilizes the Top1-dsDNA complex. However, CPT-producing plants, including Camptotheca acuminata, Ophiorrhiza pumila and Ophiorrhiza liukiuensis, are highly resistant to CPT because they possess point-mutated Top1. Here, the adaptive convergent evolution is reported between CPT production ability and mutations in their Top1, as a universal resistance mechanism found in all tested CPT-producing plants. This includes Nothapodytes nimmoniana, one of the major sources of CPT. To obtain a structural insight of the resistance mechanism, molecular dynamics simulations of CPT– resistant and –sensitive plant Top1s complexed with dsDNA and topotecan (a CPT derivative) were performed, these being compared to that for the CPT-sensitive human Top1. As a result, two mutations, Val617Gly and Asp710Gly, were identified in O. pumila Top1 and C. acuminata Top1, respectively. The substitutions at these two positions, surprisingly, are the same as those found in a CPT derivative-resistant human colon adenocarcinoma cell line. The results also demonstrated an increased linker flexibility of the CPT–resistant Top1, providing an additional explanation for the resistance mechanism found in CPT-producing plants. These mutations could reflect the long evolutionary adaptation of CPT-producing plant Top1s to confer a higher degree of resistance. Ó 2015 Elsevier Ltd. All rights reserved.

1. Introduction Eukaryotic DNA topoisomerase I (Top1) is an essential enzyme in cellular regulation and development that modifies the double stranded (ds)DNA topology via forming single-strand breaks to remove the supercoiling. Its structure is highly conserved among human, yeasts and plants. However, yeast can survive without Top1 because other topoisomerases can probably replace this function (Trash et al., 1984; Uemura and Yanagida, 1984). Being a target for anticancer drugs such as camptothecin (1) (CPT) (Fig. 1), ⇑ Corresponding authors at: Department of Biochemistry, Faculty of Science, Chulalongkorn University, 254 Phayathai Road, Bangkok 10300, Thailand. Tel.: +66 2 218 5416; fax: +66 2 218 5418 (S. Sirikantaramas). Department of Molecular Biology and Biotechnology, Graduate School of Pharmaceutical Sciences, Chiba University, Inohana 1-8-1, Chuo-ku, Chiba 260-8675, Japan. Tel.: +81 43 226 2931; fax: +81 43 226 2932 (K. Saito). E-mail addresses: [email protected] (S. Sirikantaramas), ksaito@faculty. chiba-u.jp (K. Saito).

human Top1 (HsTop1) has been extensively studied and its cocrystal structure with dsDNA has been determined (Redinbo et al., 1998). CPT (1) stabilizes a covalent Top1-DNA intermediate by blocking the rejoining step of the cleavage/religation resulting in lethal dsDNA lesions. The enzyme is composed of four different domains: the N-terminal, core, linker and C-terminal domains. The N-terminal domain is poorly conserved and dispensable for relaxation activity in vitro. The core domain is highly conserved and contains all of the catalytic residues, except for the active site tyrosine at position 723 (numbered according to HsTop1). The highly conserved C-terminal domain connects to the core domain via the short and poorly conserved linker domain. Plant Top1s have been shown to share this highly conserved structure and all the catalytic residues (Balestrazzi et al., 1996; Kieber et al., 1992). Several mechanisms of CPT (1) resistance have been reported. These include Top1 mutations (Rasheed and Rubin, 2003), increased efflux of CPT (1) by several drug transporters (Tian et al., 2005), and enhanced DNA repair (Park et al., 2002).

http://dx.doi.org/10.1016/j.phytochem.2015.02.012 0031-9422/Ó 2015 Elsevier Ltd. All rights reserved.

Please cite this article in press as: Sirikantaramas, S., et al. Structural insight of DNA topoisomerases I from camptothecin-producing plants revealed by molecular dynamics simulations. Phytochemistry (2015), http://dx.doi.org/10.1016/j.phytochem.2015.02.012

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Mutations in HsTop1 that confer CPT (1) resistance are a major problem in chemotherapy based cancer treatment. The amino acid residues affecting either the CPT-binding efficiency or the enzyme activity have been identified (Fujimori et al., 1995; Tsurutani et al., 2002; Urasaki et al., 2001). In addition, mutations in Top1 have also been reported as a self-resistance mechanism in CPT-producing plants (Sirikantaramas et al., 2009, 2008). Interestingly, one of the mutations in Top1, Asn722Ser (numbered according to HsTop1), has been found in CPT-resistant human cancer cells (Fujimori et al., 1995) and the CPT-producing plants Camptotheca acuminata, Ophiorrhiza pumila and Ophiorrhiza liukiuensis (Sirikantaramas et al., 2008). These studies can be exploited to predict the possible mutations that might occur in CPT-resistant human cancer patients in the future. The involvement of several amino acid residues in CPT (1) binding to Top1 from CPT-producing plants was previously demonstrated by using an in vivo CPT (1) toxicity assay in the Saccharomyces cerevisiae strain RS190 yeast mutant (Sirikantaramas et al., 2008). Two single mutations (Leu530Ile and Asn722Ser) in O. pumila Top1 (OpTop1) each give CPT (1) resistance, but the double mutant still resulted in partial CPT (1) resistance. In addition, the non CPT-producing Ophiorrhiza japonica also showed partial CPT (1) resistance. These results suggested the involvement of additional residues in CPT (1) resistance. Subsequently, several studies have investigated Top1 amino acid sequences in different organisms to further understand the CPT (1) and Top1 interaction. These have included other species in the Ophiorrhiza genus (Viraporn et al., 2011), the endophytic fungus, Fusarium solani, associated with C. acuminata (Kursari et al., 2011), and the insect pest, Spodoptera exigua (Zhang et al., 2013). In addition, molecular dynamics simulations (MDSs) have

Fig. 1. Structures of the compounds mentioned in this study, CPT (1), TPT (2), yohimbinic acid (3) and isorauhimbinic acid (4).

proven useful in providing the structural insight into Top1s complexed with dsDNA and topotecan (2) (TPT) (Fig. 1), a CPT derivative (Coletta and Desideri, 2013; Mancini et al., 2010; Siu and Pommier, 2013). In this study, an additional Top1 sequence was obtained from another CPT-producing plant, Nothapodytes nimmoniana (earlier known as Nothapodytes foetida), and it was concluded that the mutations in Top1 are a common CPT (1) resistance mechanism in all CPT-producing plants. MDSs were also performed to demonstrate the structure of plant Top1s complexed with dsDNA and TPT (2), and additional amino acid substitutions were identified conferring CPT (1) resistance.

2. Results and discussion 2.1. Point mutation in N. nimmoniana Top1 (NnTop1) confers CPTresistance To demonstrate that point mutations in Top1 are a universal self-resistance mechanism among all CPT-producing plants known to date, Top1 was cloned from N. nimmoniana by PCR-based cloning using the same degenerate PCR primers as previously reported (Sirikantaramas et al., 2008). Then 50 - and 30 -RACE were performed to obtain the full-length cDNA. By comparison with other reported residues involved in direct/indirect CPT-binding, two amino acid substitutions (Asn421Lys and Leu530Ile) correlated with (and so potentially conferred) CPT resistance (Fig. 2). These were identical to the mutations found in CaTop1 and OpTop1, respectively. NnTop1 resistance to CPT (1) was confirmed by conducting an in vivo assay in the Top1-deleted S. cerevisiae strain RS190 (Eng et al., 1988; Sirikantaramas et al., 2008). In the glucose-containing medium (repressed condition), CPT (1) did not affect the growth of the yeast containing the expression vector of either NnTop1 or CrTop1 (Fig. 3). After induction of NnTop1 with galactose, yeast cells were able to grow in the presence of CPT (1) while yeasts expressing the CPT-sensitive Top1 (Catharunthus roseus Top1, CrTop1) could not grow in the medium containing CPT (1) (Fig. 3), supporting the CPT-resistance of NnTop1. Although protein expression level of NnTop1 was not confirmed, a previous study showed that various plant Top1s can be successfully overexpressed in yeast cells (Sirikantaramas et al., 2008). The phylogenetic analysis of NnTop1, together with Top1s from other plants and human, indicated that Top1s from either CPT-producing or non-producing plants are mixed together (Fig. 4). Despite the distant relationship of the CPT-producing Ophiorrhiza spp., C. acuminata and N. nimmoniana, they shared identical amino acid substitutions linked to (conferring) CPT-resistance, suggesting they may have arisen by convergent evolution from the need for CPTresistance.

Fig. 2. Amino acid substitutions in the Top1s from selected CPT-producing plants and non-producing organisms. Shown are the amino acids that are involved in Top1 catalysis and CPT direct/indirect binding. The numbering is based on HsTop1. Double underlined characters indicate residues that have been previously reported (Sirikantaramas et al., 2008), while single underlined characters indicate residues mentioned in this study. The symbols ‘‘+’’ or ‘‘’’ indicate a CPT-producing or not organism, respectively.

Please cite this article in press as: Sirikantaramas, S., et al. Structural insight of DNA topoisomerases I from camptothecin-producing plants revealed by molecular dynamics simulations. Phytochemistry (2015), http://dx.doi.org/10.1016/j.phytochem.2015.02.012

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Fig. 3. In vivo CPT (1) sensitivity assay in Top1-deleted Saccharomyces cerevisiae strain RS190. Ten-fold serial dilutions (indicated by triangles) of exponentially growing yeast cells transformed with either NnTop1 or CrTop1 were spotted (at 5 lL) onto selective media in the presence of galactose or galactose plus 5 lg/mL CPT. Galactose was used to induce expression of exogenous Top1s.

Fig. 4. NJ based phylogenetic tree of Top1s from plants and human. The symbols ‘‘+’’ or ‘‘’’ indicate a CPT-producing or not organism, respectively. The numbers on each branch represent percent bootstrap values (1000 replicates). The scale bar represents a genetic distance of 0.1 amino acid substitution per position. GenBank accession codes for all these sequences are given in the text.

Interestingly, Rauvolfia serpentina has been reported to produce two other types of indole alkaloids (yohimbinic acid (3) and isorauhimbinic acid (4)) (Fig. 1) that exhibit HsTop1 inhibitory activity (Itoh et al., 2005). The amino acid sequence of R. serpentina Top1 (RsTop1) was thus investigated, but no mutations were observed in positions reported to be involved in CPT (1) resistance (Fig. 2). This could suggest either a different CPT-resistance mechanism or a different interaction between RsTop1 and yohimbinic acid (3)/isorauhimbinic acid (4). It is unlikely that the intracellular transport/accumulation of these two compounds would be different from that previously reported for CPT (Sirikantaramas et al., 2007). 2.2. Additional residues involved in CPT resistance revealed by MDSs Previously, Ile530 and Ser722 in O. pumila and Lys421 in C. acuminata were identified as being involved in CPT (1) resistance (Sirikantaramas et al., 2008). However, many more amino acids seem to be involved in the resistance mechanism. The double Ile530Leu: Ser722Asn mutation found in the CPT-resistant OpTop1 still exhibited CPT (1) resistance (Sirikantaramas et al., 2008). Several studies employed the combination between experimental and simulative approaches to reveal CPT (1) resistance mechanisms (D’Annessa et al., 2013; Fiorani et al., 2009). MDSs were therefore, performed on the ternary Top1/DNA/TPT complex to identify additional amino acid(s) that might be involved. To determine the stability of the whole structure of each of the different HsTop1, CrTop1 and OpTop1 models complexed to dsDNA and TPT (2), the root-mean-square displacement (RMSD) of each system was calculated (Fig. 5). The HsTop1 complex, which was used as the template to build the other complex models, reached equilibrium much earlier than the other three plant Top1 complexes. How-

Fig. 5. Illustration of the root-mean-square displacements (RMSDs). RMSDs of all heavy atoms for each domain of Top1s from human (HsTop1), C. roseus (CrTop1) and O. pumila (OpTop1) are indicated for the N-terminal (black), core (red) linker (green) and C-terminal (blue) domains.

ever, all the complexes reached equilibrium within 15 ns and so in each case the last 15 ns of the 30 ns MDS was used (Fig. 5). The average structures over the last 15-ns MDS trajectories gave a significant difference in the orientation of the OpTop1 linker domain, but only a slight difference in that of the CrTop1 linker domain. The superimposition of the simulated HsTop1 onto the other models suggests a difference in the flexibility of the linker domain between the CPT-sensitive and -resistant Top1 (Fig. 6). In fact, the flexibility of the linker domain has previously been reported to confer CPT (1) resistance in HsTop1 (Fiorani et al., 2003). Accordingly, the amino acid residues within and close to the linker region were investigated, since it can be hypothesized that mutation(s) in amino acid residues in those regions of OpTop1 would disrupt the interaction between the linker domain and other residues possibly residing at the core domain helix (so-called helix 17), which is located close to the linker domain (Fig. 6). At least five residues (Leu617, Arg621, Arg624, Asp707 and Glu710) in HsTop1 have been reported to be involved in forming the interaction between the helix 17 and the linker domain through several hydrophobic residues and salt bridges (Chillemi et al., 2008; Tesauro et al., 2013). Here, the MDS results showed that the side-chains of these five residues were in close contact in both the CPT-sensitive HsTop1 and CrTop1, making those interactions possible (Fig. 7A and B). In contrast, the average simulated structure of the CPT-resistant OpTop1 showed no evidence of the interaction between helix 17 and the linker domain (Fig. 7C). Multiple alignment of the amino acid sequences for Top1s (Supplementary Fig. 1) together with the homology-modeled structures of OpTop1 and CaTop1 were used to identify what amino acid residues are uniquely found in the CPT-producing plants and are

Please cite this article in press as: Sirikantaramas, S., et al. Structural insight of DNA topoisomerases I from camptothecin-producing plants revealed by molecular dynamics simulations. Phytochemistry (2015), http://dx.doi.org/10.1016/j.phytochem.2015.02.012

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Fig. 6. Structural superimposition of the average conformation generated during the last 15 ns MDS snapshots of each system. Superposition of HsTop1 (green) and (A) CrTop1 (blue) or (B) OpTop1 (red) in a complex with dsDNA and TPT (2).

Fig. 7. Close-up average structures during the last 15 ns MDS snapshots between the helix 17 and linker domains. The amino acid side-chains possibly involved in the interaction between helix 17 (left) and linker domain (right) of (A) HsTop1, (B) CrTop1 and (C) OpTop1 are depicted. Amino acid residue numbering is based on HsTop1.

residues at these two positions would disrupt the interactions and possibly affect the linker flexibility, which could reduce the affinity between Top1 and CPT (1) (Fig. 7). Fiorani et al. (2003) have shown that only the single Ala653Pro mutation in the linker domain can increase the linker flexibility, resulting in an altered conformation during the drug binding to the enzyme. Interestingly, the Glu710Gly mutant in HsTop1 exhibited a high religation rate in vitro (Tesauro et al., 2013). Conferring CPT (1) resistance can be explained as that the increased religation rate hardly affects its binding. However, this observation has to be experimentally confirmed for these plant Top1s. Fig. 8. Top1 homology modeled structures showing a close up view of the amino acid residues in helix 17 and the linker domain. (A) HsTop1, (B) OpTop1 and (C) CaTop1. Mutated amino acids identified in this study are shown in red.

2.3. Multiple point mutations in plant CPT-resistant Top1s

possibly involved in the linker flexibility. From this approach, Gly617 in the helix 17 of OpTop1 and Gly710 in the CaTop1 linker domain that possibly confers CPT (1) resistance were identified (Figs. 2 and 8). Gly617 is also present in all Top1s from other CPT-producing Ophiorrhiza species except for that from Ophiorrhiza ridleyana. In O. ridleyana Top1, the amino acid at this position was replaced with Ala (Supplementary Fig. 1). However, substitution with either Gly or Ala may produce a similar result. Surprisingly, the mutations at Leu617Ile and Glu710Gly in HsTop1 have recently been found in HCT116 colon adenocarcinoma cell lines that are resistant to SN38, a CPT (1) derivative (Gongora et al., 2011). Consequently, the mutations of CPT-producing plant Top1s to Gly

Previous studies have identified several mutations involved in CPT (1) resistance, where Asn421Lys, Le530Ile and Asn722Ser have been demonstrated to confer resistance using the in vivo yeast cell viability assay (Sirikantaramas et al., 2008). Viraporn et al. (2011) pointed out that Gly717Ser is restrictedly found in all CPT-producing Ophiorrhiza Top1s. However, the involvement of this mutation in CPT (1) resistance has not been evaluated. Mutating the yeast Top1 Gly721 (analogous to the HsTop1 Gly717) to Asp, Glu, Asn, Gln, Leu or Ala increased the Top1 sensitivity to CPT (1), whereas mutating it to Val or Phe increased the resistance level (Van der Merwe and Bjornsti, 2008). This residue is located at the juncture between the linker and C-terminal domains and has been proposed to facilitate the linker dynamics that accompany CPT (1) binding.

Please cite this article in press as: Sirikantaramas, S., et al. Structural insight of DNA topoisomerases I from camptothecin-producing plants revealed by molecular dynamics simulations. Phytochemistry (2015), http://dx.doi.org/10.1016/j.phytochem.2015.02.012

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However, the question remains whether mutating Gly to Ser would enhance CPT (1) resistance. In order to investigate the effect of Gly717Ser in OpTop1, computational alanine scanning mutagenesis was employed and the influence on the affinity for CPT was analyzed. A variety of single mutants of HsTop1, CrTop1 and OpTop1 were generated by mutating residues close to either the Top1 active site or the CPT (1) binding site to Ala (Supplementary Table 1). Significant changes in the relative influence of the mutation on the binding free energy were observed when mutating Arg364, Lys532 and Asp533 to Ala in all the examined mutated Top1s. These three residues stabilized TPT (2) (Supplemental Fig. 2) due to direct contact with the drug (Staker et al., 2002). Arg364 and Asp533 stabilized the TPT (2) binding via hydrogen bond formation in HsTop1 and CrTop1, whilst hydrogen bond with Lys532 was only observed in OpTop1 (Supplemental Fig. 3), resulting in a lengthened distance with 1T-O2 (3.2 Å while 2.0 and 2.7 Å in CrTop1 and HsTop1, respectively). In addition, the individual contribution of phosphotyrosine (PTyr723) apparently decreased by 2.5 kcal/mol in OpTop1 complex compared to the others. Reduction in drug–enzyme interactions might be affected from the mutation(s). Noticeably, mutating other residues that are known to indirectly contact with the drug did not show any significant differences in relative binding free energy. This included OpTop1: Ser717Ala. These results suggest that computational alanine scanning mutagenesis is a potentially useful method for analyzing residues that are directly involved in ligand binding. Although this method did not confirm the role of Ser717 in OpTop1, the possibility that a mutation at this position could confer CPT (1) resistance cannot be ruled out. It has been clearly shown that CPT-producing plants possess several point mutations in Top1s that might be required for a high degree of CPT (1) resistance. The double Ile530Leu and Ser722Asn mutant of OpTop1 has previously been shown to be sensitive to CPT (1) (Sirikantaramas et al., 2008). Mutations of the additional residue(s) identified in this study would then potentially result in a highly CPT-sensitive OpTop1. So far, only two point mutations have been addressed in NnTop1 in this study. It is possible that there are hidden mutated residues that are responsible for CPT (1) resistance. Identification of substituted amino acids uniquely found in CPT-resistant Top1 would lead to the discovery of additional point mutations that confer CPT (1) resistance. 3. Concluding remarks In conclusion, in addition to the three amino acids previously identified in CPT-producing plants (Asn421Lys, Leu530Ile and Asn722Ser) nearby the CPT (1) binding site, two new mutations identified by MDSs (Leu617Gly and Asp710Gly) are reported that could affect the linker domain flexibility and result in CPT (1) resistance. 4. Experimental 4.1. Plant materials Young leaves of N. nimmoniana were collected in Medicinal Plant Gardens of the Graduate School of Pharmaceutical Sciences at Chiba University, Japan and used for RNA extraction. 4.2. cDNA cloning The cDNAs encoding NnTop1 were cloned as previously reported (Sirikantaramas et al., 2008) using SMART RACE kit (Clontech). Other primers used to clone this gene are as follows: 50 RACE, 50 GAAATACTTTAGCTGTGAGGCCTGG-30 ; 30 RACE, 50 -ATGCTAGATAC-

GAGTAAACTGAAT-30 Full-length/F, 50 -AAAAAGCAGGCTTAAGAAAG GATGGATAGTAGTGACGA-30 ;

Full-length/R,

50 -AGAAAGCTGGG-

0

TATTAGAATCTAAAGCTGGGATCAACA-3 . The regions of primers that introduced the Gateway attB1 forward or attB2 reverse primers are underlined. The nucleotide sequence of NnTop1 has been deposited in the DNA Databank of Japan (http://www.ddbj.nig.ac. jp) under accession number AB927719. 4.3. Yeast expression and CPT sensitivity assay NnTop1 and CrTop1 were separately cloned into the Gateway expression vector pYES-DEST52 and the respective constructs were transformed into S. cerevisiae strain RS190 (MATa, Top1D) purchased from the American Type Culture Collection. CPT (1) sensitivity assay was conducted according to Sirikantaramas et al. (2008) individual transformants were grown in a selective medium containing glucose. The overnight cultures were adjusted to an OD600 of 0.3 and serially diluted 10-fold, then 5 lL each were spotted onto selective plates supplemented with 2% (w/v) glucose or galactose and 0 or 5 lg/mL of CPT (1) dissolved in DMSO. All plates were adjusted to contain a final DMSO concentration of 0.25% (v/v). 4.4. Phylogenetic analysis of Top1 The amino acid sequences encoding Top1 were aligned with ClustalW, implemented in the MEGA version 5.10 program (Tamura et al., 2011), using the default parameters, and then used to construct a phylogenetic tree using the neighbor-joining (NJ) distance-based method under the default parameters of the same software. Bootstrap values were statistically calculated from 1000 replicates. The amino acid sequences of Top1s were retrieved from the National Center for Biotechnology Information GenBank database for those from O. fucosa (accession number BAK32956), O. pumila (BAG31373), Ophiorrhiza sp. 35 (BAK32963), Ophiorrhiza harrisiana (BAK32957), O. ridleyana (BAK32961), O. liukiuensis (BAG31374), Ophiorrhiza plumbea (BAK32959), Ophiorrhiza pedenculata (BAK32958), Ophiorrhiza trichocarpon (BAK32962), Ophiorrhiza pseudofasciculata (BAK32960), O. japonica (BAG31375), C. roseus (BAG31377), C. acuminata (BAG31376), Arabidopsis thaliana (CAA40763) and Zea mays (NP001157806). In addition, that from Rauvolfia serpentina was retrieved from the Medicinal Plant Genomic Resource (http://medicinalplantgenomics.msu.edu) using the BLAST search tool of RNA-Seq data. 4.5. Molecular modeling The reported X-ray crystal structure of HsTop1 covalently bound to dsDNA and complexed with TPT (2) (Staker et al., 2002), taken from the Protein Data Bank entry code 1K4T, was used as the template structure for building the model structures of CrTop1 and OpTop1. The crystal structure 1K4T contains two conformations for TPT (2); one conformation captures the situation that the pyranone moiety of TPT (2) is dissociated by hydrolysis (carboxylate form) and the other shows no hydrolysis for pyranone moiety (lactone form). The latter conformation was selected for TPT (2) in the present computation model. The homology modeling module implemented in Discovery Studio 2.5Accerys Inc was employed to build the CrTop1 and OpTop1 structures. Afterwards, the dsDNA and TPT (2) were superimposed into the DNA binding groove and the active site of each model structure. 4.6. Molecular dynamics simulations (MDSs) Three kinds of the ternary Top1/DNA/TPT (2) complexes were prepared for MDSs, which were performed with the AMBER10

Please cite this article in press as: Sirikantaramas, S., et al. Structural insight of DNA topoisomerases I from camptothecin-producing plants revealed by molecular dynamics simulations. Phytochemistry (2015), http://dx.doi.org/10.1016/j.phytochem.2015.02.012

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package program (Case et al., 2008), as previously reported (Khuntawee et al., 2012; Rungrotmongkol et al., 2010). The AMBER ff03 force field (Duan et al., 2003) was used as the parameters for proteins and ions. Meanwhile, the general AMBER force field (GAFF) (Wang et al., 2004) and AMBER ff03 force field were used as the parameters for TPT (2) using parmchk program. In order to obtain the partial atomic charges of the compound, the stable structure of the inhibitor was determined through the geometry optimization at the HF/6-31g(d) level of theory with Gaussian03 program (Frisch et al., 2004). Subsequently, the electrostatic potential (ESP) charges were calculated with the same method and basis set, followed by the restrained electrostatic potential (RESP) fitting (Cieplak et al., 1995). The protonation states of all ionizable amino acid residues (Arg, Lys, Asp and Glu) were set to those of each respective single amino acid at pH 7, whereas the hydrogen bond interactions with the surrounding residues were considered for the determination of the protonation state of His. All missing residues were generated by the Discovery Studio. Hydrogen atoms were subsequently added using the LeaP module in AMBER10. The complex models were minimized with 1000 steps of steepest descent (SD) followed by 2000 steps of conjugated gradient (CG) to reduce the bad contact and steric hindrance. Each complex was then embedded in a cubic box solvated with TIP3P water (Jorgensen et al., 1983), keeping a minimum distance of 10 Å from the protein surface to the wall of the box. Sodium ions were introduced to neutralize the charged Top1 protein and dsDNA. Water molecules were minimized with 1500 steps of SD minimization, followed by another 1500 steps of CG one, while Top1, dsDNA and TPT (2) were restrained with a force constant of 500 kcal/mol Å2. Finally, the whole system was fully optimized with 1500 steps of SD and CG minimization. MDS was performed for each Top1/dsDNA/TPT complex with the periodic boundary in the NPT ensemble condition at 1 atm. The SHAKE algorithm (Ryckaert et al., 1977) was employed to constrain all covalent bonds involving hydrogen atoms. A cutoff distance of 10 Å and the particle mesh Ewald method (Essmann et al., 1995; Ryckaert et al., 1977) were applied for calculating non-bonded interactions and long-range electrostatic interactions, respectively. MDSs were performed for 30 ns as 0.2 ns of heating to 310 K, 2.8 ns of restrained run, 12 ns of equilibration and 15 ns of production run with a time step of 2 fs. Finally, the MD trajectories of the production phase were taken for analysis. Note that the rootmean-square displacement (RMSD) and per-residue decomposition energy including its components were investigated using the ptraj and MM/GBSA modules. 4.7. Computational alanine scanning mutagenesis Computational alanine scanning mutagenesis is widely used to find the hot spot residues that importantly contribute to proteinprotein/ligand binding. Alanine scanning was executed by calculating the difference in binding free energies (DDG) between the wild type (DGWT) and mutant type (DGMT) based on the molecular mechanics/Poisson Boltzmann surface area (MM/PBSA) method. To clarify the contribution of the respective residues to the intermolecular interaction, the internal dielectric constants of 2.0 for nonpolar amino acids, 3.0 for polar amino acids and 4.0 for charged residues including His to clarify the contribution of the respective residues to the inter-molecular interaction (Moreira et al., 2007, 2006). Acknowledgements The authors thank Dr. Robert Butcher and Mr. Surachat Tangpranomkorn, Faculty of Science, Chulalongkorn University, for editing the manuscript. Parts of this research were supported

by Grant-in-Aids from The Ministry of Education, Culture, Sports, Science and Technology (MEXT) and the Japan Society for the Promotion of Science (JSPS) (to M.Y. and K.S.), and Special Task Force for Activating Research (STAR), Ratchadaphiseksomphot Endowment Fund of Chulalongkorn University 56-007-23-004 and Thailand Research Fund IRG578008 (to S.S.). A.M. thanks the Ratchadaphiseksomphot Endowment Fund from Chulalongkorn University for postdoctoral fellowship. The Computational Chemistry Unit Cell at Chulalongkorn University provided the computing facilities and resources.

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