Báo cáo khoa học: The bI/bIII-tubulin isoforms and their complexes with antimitotic agents pptx

In this study, we use a com-bination of molecular modeling, docking and molecular dynamics techniques to investigate the molecular basis of paclitaxel resistance and IDN5390 sensitivity,

antimitotic agents

Docking and molecular dynamics studies

Matteo Magnani1, Francesco Ortuso2, Simonetta Soro3, Stefano Alcaro2, Anna Tramontano3

and Maurizio Botta1

1 Dipartimento Farmaco Chimico Tecnologico, Universita` degli Studi di Siena, Italy

2 Dipartimento di Scienze Farmacobiologiche ‘Complesso Nin’ Barbieri’ Universita` degli Studi di Catanzaro ‘Magna Graecia’, Roccelletta di Borgia (CZ), Italy

3 Dipartimento di Scienze Biochimiche ‘A Rossi Fanelli’, Universita` degli Studi ‘La Sapienza’, Rome, Italy

Microtubules are filamentous dynamic polymers

com-posed of a⁄ b-tubulin heterodimers involved in a

diverse range of cellular functions including motility,

morphogenesis, intracellular trafficking of

macromole-cules and organelles, and mitosis and meiosis [1,2]

The role played by microtubules in the cell division

process makes them attractive targets for anticancer

therapy [3], a perspective that has been explored by

using tubulin-binding agents [4] These compounds are

able to disrupt microtubule dynamics and can act

either as microtubule destabilizers (such as vinca

alka-loids) or as microtubule stabilizers (such as taxanes)

Among the latter, paclitaxel (Fig 1 left) has been

dem-onstrated to be effective for the treatment of ovarian,

breast, and nonsmall cell lung carcinomas [5] These molecules have the drawback of being scarcely select-ive, but an even more significant problem that limits their usage in the treatment of malignancies is the emergence of resistance There are essentially two routes to resistance [4]: (i) expression of the P-glyco-protein [6], which is able to pump the antitumoral compounds out of the tumor cell; and (ii) emergence

of structural modification of the microtubules them-selves, both via mutations and modifications of their isotype composition, in particular that of their b-sub-unit [7–9] In humans, seven isoforms of b-tubulin, displaying different patterns of tissue expression, have been identified [10,11] In particular, bI is

Keywords

docking; epothilone A; IDN5390; paclitaxel;

tubulin

Correspondence

M Botta, Dipartimento Farmaco Chimico

Tecnologico, Universita` degli Studi di Siena,

Via Alcide de Gasperi, 2, I-53100 Siena, Italy

Fax: +39 577 234333

Tel: +39 577 234306

E-mail: botta@unisi.it

(Received 7 April 2006, revised 16 May

2006, accepted 23 May 2006)

doi:10.1111/j.1742-4658.2006.05340.x

Both microtubule destabilizer and stabilizer agents are important molecules

in anticancer therapy In particular, paclitaxel has been demonstrated to be effective for the treatment of ovarian, breast, and nonsmall cell lung carci-nomas It has been shown that emergence of resistance against this agent correlates with an increase in the relative abundance of tubulin isoform bIII and that the more recently discovered IDN5390 can be effectively used once resistance has emerged In this paper, we analyze the binding modes

of these antimitotic agents to type I and III isoforms of b-tubulin by com-putational methods Our results are able to provide a molecular explan-ation of the experimental data Using the same protocol, we could also show that no preference for any of the two isoforms can be detected for epothilone A, a potentially very interesting drug for which no data about the emergence of resistance is currently available Our analysis provides structural insights about the recognition mode and the stabilization mech-anism of these antimitotic agents and provides useful suggestions for the design of more potent and selective antimitotic agents

Abbreviation

PDB, Protein Data Bank.

constitutively expressed and represents, in general, the

most abundant isotype, whereas bIII expression is

restricted to neuronal tissues and testis There are

sev-eral studies reporting that an increase in the relative

abundance of isoform III destabilizes the microtubules

[12,13] and clear indications that it correlates with

paclitaxel resistance, both in vitro and in vivo [14–18]

Recent studies have shown that a seco-taxan (IDN5390,

Fig 1 middle) [19], although less potent than paclitaxel,

is active on tumor cells overexpressing isoform III and

therefore could be used in cases where resistance to

paclitaxel has emerged [20] In this study, we use a

com-bination of molecular modeling, docking and molecular

dynamics techniques to investigate the molecular basis

of paclitaxel resistance and IDN5390 sensitivity,

through the analysis of the complexes between these two

ligands and the isotypes I and III of the human

b-tubu-lin We also investigated the complexes involving the bI

and bIII isoforms with epothilone A (Fig 1 right)

Epo-thilones are microtubule stabilizing agents, sharing a

common mechanism of action with taxanes [21] To the

best of our knowledge, no data is available about the

activity of this class of compounds on different isoforms

of tubulin, even though these molecules are gaining

more and more attention in antitumoral therapy [22]

Results

Analysis of tubulin crystallographic models and

docking with the three ligands

The structures of ligands used in this study are

repor-ted in Fig 1; with respect to paclitaxel and IDN5390,

epothilone A is characterized by a less complex

molecular structure There is some confusion in the

lit-erature and in databases about the nomenclature of

the various tubulin isoforms The bI and bIII genes

have been recently re-sequenced [17], and we noticed

that the protein annotated as tubulin bII chain (Code:

TBB2_HUMAN, P07437) in SwissProt corresponds to

the sequence of the tubulin bI chain We also checked that the confusion did not reflect population polymor-phisms: no single nucleotide polymorphism is reported

in the human genome in positions that are different between bI and bIII The problem has now been brought to the attention of the database curators There are several structural determinations of the tubulin dimer from different sources, some of which have been obtained by binding tubulin to a zinc sheet

in order to obtain a bidimensional crystal, some others

by fitting the zinc sheet structures in electron microscopy data, some by X-ray diffraction of crystals containing

a tubulin dimer in complex with small ligands and⁄ or other proteins, and some by modeling No structure determination is available for the human proteins and therefore we needed to build comparative models for the human proteins In particular, we concentrated on isoforms I and III of the human tubulin b subunit, as this subunit hosts the common binding site for our molecules of interest [23,24]

The sequence identity between tubulin from different sources and their human counterpart is very high (between 89 and 94%), nevertheless it is very difficult

to assess which of the available structural determina-tions better reflects the conformation of the protein in physiological conditions and is therefore better suitable

to be used as template in the model-building proce-dure In order to select the appropriate template, we analyzed all tubulin-related entries in the Protein Data Bank (PDB) and performed an all-against-all compar-ison of the complete structures and of the most rele-vant parts of the structures (dimer interface and binding site) The differences in the overall structure are rather high in terms of rmsd (up to 4 A˚ and more, see supplementary material, Table S1) More limited is the structural variation of the set of residues involved

in the interaction with paclitaxel and epothilone A, which are relevant for our purposes (see supplementary material, Table S2) The variability seems to be mostly correlated with the nature of the ligand and,

conse-Fig 1 Chemical structures of paclitaxel (1), IDN5390 (2) and epothilone A (3).

quently, the best choice seemed to be to select as

tem-plates the structures bound to the ligands that we

planned to study and we used 1JFF as template to

build our comparative model, named hTUB We used

a standard comparative modeling procedure, replacing

the residues of the template with those of the target

according to the sequence alignment obtained using

clustalw[25] with standard parameters The replaced

sidechains were positioned in their most commonly

observed conformation [26] and the model was

opti-mized using 100 cycles of steepest descent energy

mini-mization using discover [27] and the CVFF force

field For the initial positioning of the paclitaxel

moi-ety in the complex, we used the orientation found in

the 1JFF structure, which contains a dimer of tubulin

complexed with paclitaxel [23], to position the ligand

in hTUB In the case of IDN5390, the crystallographic

structure of its complex with b-tubulin is not available

Consequently, the ligand was docked into the same

binding site of paclitaxel (and epothilone A), assuming

that these two very similar molecules act in a similar

fashion As a result of docking (for details, see

Experi-mental procedures), IDN5390 was located within the

binding site in a conformation which closely resembles

that of paclitaxel, with the macrocyclic moiety and the

lateral chains occupying the same regions of the pocket

(Fig 2A,B) For simulations involving epothilone, we

took advantage of the availability of the 1TVK

struc-ture, which contains the structure of a tubulin dimer

complexed with epothilone A [24] 1TVK was

superim-posed to hTUB and the ligand positioned in the

con-text of the model structure in the same relative

orientation as observed in 1TVK (Fig 2C) Thus, a

common tubulin structure (hTUB) was used for all

three ligands under analysis (Fig 3), in an attempt to

limit the biases that could derive from using different

starting protein structures The procedures described

above were followed to obtain the starting structures

of the six complexes of paclitaxel, IDN5390 and

epo-thilone A with both bI and bIII isotypes of tubulin

Molecular dynamics and thermodynamics

calculations

The starting complexes, built as described above, were

analyzed by means of molecular dynamics Such

analy-sis involved, at first, paclitaxel and IDN5390, for

which data about the activity towards microtubules

with different composition in terms of b-tubulin

iso-types are reported, and was subsequently extended to

epothilone A, for which to date no data is available

Human tubulin bI and bIII isoforms complexed with

paclitaxel (referred to as P1 and P3, respectively), and

A

B

C

Fig 2 Location of ligands within the hTUB binding site (solvent-accessible surface representation) in the starting complexes (A) paclitaxel, (B) IDN5390, and (C) epothilone A Nonpolar hydrogen atoms of the ligands are not shown.

with IDN5390 (referred to as I1 and I3, respectively),

were first energy minimized and then subjected to

backbone-constrained molecular dynamics In the

course of each simulation, 100 ligand–protein

com-plexes were sampled at regular time intervals and were

fully minimized A conformational clustering analysis

was applied to the resulting structures Representative

structures for the whole set of collected and optimized

frames were selected by performing a Boltzmann

ana-lysis, in order to take into account both the energy

and the number of structures within each cluster The

reduced number of selected structures was used to

investigate the molecular basis of the difference in the

calculated binding energies of both ligands for the bI

to bIII-tubulin isoform Finally, in all simulations, the

average drug–protein binding energies (DG-, DH- and

DS-values) were computed according to the MOLINE

methodology reported by some of us [28] The results

are reported in Table 1 The data predict a higher

affinity of paclitaxel for the bI isoform than for the

bIII isoform, and an opposite behavior of IDN5390

This is in good agreement with experimental data, as

we are able to correctly reproduce the differences in sensitivity to paclitaxel and IDN5390 observed for microtubules with different isotype composition Figure 4 shows the region around the ligand for both the P1 and P3 complexes, in one of the representative sampled structures (for other representative structures, quite similar considerations can be made) The binding

of paclitaxel to bI-tubulin (Fig 4A) involves both hydrogen bonds and multiple hydrophobic contacts; most interactions are in agreement with the crystallo-graphic structure of the complex (taken as starting structure) and have already been described [22,29,30] (supplementary material, Table S3) In particular, the C2 phenyl ring is involved in hydrophobic interactions with Leu217, His229 and Leu230, while the C4 acetate makes hydrophobic contacts with Phe272, Pro274 and Leu371 Two hydrogen bonds are established between the oxygen of the oxetane ring and the NH backbone

of Thr276, and between the C2¢ hydroxyl group and the NH backbone of Gly370 However, as expected,

Fig 3 Ribbon representation of the main secondary structure

ele-ments characterizing the b-tubulin binding site for the ligands under

analysis: paclitaxel (green), IDN5390 (magenta), epothilone A

(orange).

Table 1 Free energy, enthalpy and entropy for the drug-protein

complexes computed at 300 K P1 and P3 refer to paclitaxel bound

to human b-tubulin isoforms I and III, respectively I1 and I3 to

IDN5390 bound to human b-tubulin isoforms I and III.

Complex

DG

(kcalÆmol)1)

DH (kcalÆmol)1)

DS (calÆmol)1)

A

B

Fig 4 Paclitaxel in complex with (A) bI-tubulin (P1), and (B) bIII-tubulin (P3) For sake of clarity, nonpolar hydrogen atoms are omit-ted and hydrogen bond interactions are represenomit-ted by black dashed lines.

the molecular dynamics simulation is also

accompan-ied by structural variations of the initial complex

Especially the flexible M-loop experiences a slight

rear-rangement that results in additional interactions with

the ligand As shown in Fig 4A, in the P1 complex

the C7 hydroxyl group of paclitaxel forms two

hydro-gen bonds with the main chain carbonyl group and

amino group of Ser277 and Gln282, respectively A

hydrogen bond interaction is also established between

the C10 acetate and the lateral chain of Arg284 Such

hydrogen bonds are stable during the course of the

whole simulation, being present in >95% of the

sampled structures Finally, in P1, paclitaxel is also

engaged in hydrogen bond interactions with Glu27

Differently from the interactions described above, the

latter hydrogen bonds are less stable: in fact, due to

the high mobility of the lateral chain of Glu27 they

are observed in most of the sampled structures only

after minimization In the complex with bIII-tubulin

(P3, Fig 4B), the ligand is characterized by a very

sim-ilar binding conformation compared with P1, while the

structure of the M-loop is somewhat different in the

two complexes, owing to the replacement of Ser277 in

isoform bI with Ala277 in isoform bIII This results in

a different and less effective interaction with the C7–

C10 moiety of the ligand In comparison with P1,

Arg278 is directed towards the inside of the binding

pocket, establishing two hydrogen bonds with carbonyl

at C9, only one of which is consistently observed in

the course of the molecular dynamics simulations

Interestingly, in P1 Ser277 forms a hydrogen bond

with Ser280, thus directing its carbonyl group towards

the C7 position of the paclitaxel ring and forming a

hydrogen bond with its OH The substitution

Ser277-Ala, present in the bIII isoform, does not allow this

interaction to take place Similarly, the different

rear-rangement of the M-loop in bIII prevents the C10

acetate from interacting with Arg284 In P1, Glu27 is

hydrogen bonded to the ligand, while in P3 this

inter-action is absent In this isoform, Glu27 interacts with

Arg320, which, in turn, forms a hydrogen bond with

Ser241 Such an interaction network cannot take place

in P1, where Ser241 is replaced by a cysteine, which

does not interact with Arg320

The models of the complexes of IDN5390 with the

bI and bIII-tubulin isoforms are shown in Fig 5 The

bound conformation of the ligand is quite similar in

the I1 and I3 complexes, in part resembling that of

paclitaxel In fact, similarly to paclitaxel, hydrophobic

interactions are established between the C2 phenyl ring

of IDN5390 and Leu217, His229 and Leu230, as well

as between the C4 acetate and Phe272, Pro274 and

Leu371 IDN5390 too is engaged in two hydrogen

bonds involving the oxetane ring and the C2¢ hydroxyl group with Thr276 and Gly370, respectively Further-more, the hydroxyl group in the C2¢ position of paclit-axel forms a second hydrogen bond with Glu27 of bI isotype, while the equivalent atom of IDN5390 is involved in a second hydrogen bond with the sidechain

of Asp26 in both isoforms However, a pattern of interactions different from those observed for paclit-axel is established with the M-loop, whose structural rearrangement is, also in this case, different in the bI and bIII isotypes In both complexes, the C1 and the C9 hydroxyl groups interact with His229 and Gln282, respectively, through hydrogen bond interactions Nev-ertheless, the different rearrangement of the M-loop in the two isoforms (due to the replacement of Ser277 in I1 with Ala277 in I3) results in some important differ-ences in the binding of IDN5390 Remarkably, only in the bIII isoform does the conformation of the M-loop allow the lateral chain of Arg278 to move towards the ligand and to favorably interact with it through hydro-gen bonds with the C1 hydroxyl group and the C2

A

B

Fig 5 IDN5390 in complex with (A) bI-tubulin (I1), and (B) bIII-tubulin (I3) For sake of clarity, nonpolar hydrogen atoms are omit-ted and hydrogen bond interactions are represenomit-ted by black dashed lines.

benzoyl chain (Fig 5B) Moreover, such location of

the sidechain of Arg278 also results in a better

‘entrap-ment’ of the ligand within the binding site compared

to isoform bI, as shown in Fig 6 As mentioned

above, in the P3 complex, Arg278 also points towards

the inside of the pocket, but it does so to a much

lower extent than in the I3 complex In fact, the

differ-ent structure of the macrocycle in paclitaxel with

respect to IDN5390 induces a different rearrangement

of the M-loop, which does not allow Arg278 to

inter-act with the ligand as closely as in the IDN5390 bound

structure and therefore the paclitaxel in bIII isotype is

less well packed within the protein structure

Taken together, our analysis of the P and I

com-plexes reveals that for both paclitaxel or IDN5390 the

different calculated binding energies can be mainly

ascribed to the replacement of Ser277 of the bI

iso-form with Ala277 in bIII Such a residue is located

within the M-loop (which constitutes an important

part of the binding pocket, as shown in Fig 3) The

importance of the role of this residue in ligand binding and in the tubulin structure has been pointed out recently [11] According to our findings, the role played by residue 277 is crucial not only because Ser277 is directly involved in the binding of paclitaxel with the bI isotype, but also because its replacement with Ala277 in bIII induces a conformational rear-rangement of the M-loop which, in turn, results in dif-ferent interactions of the ligands with other residues in the M-loop In particular, paclitaxel interacts through hydrogen bonds with Ser277, Gln282 and Arg284 in the case of the bI isoform and only with Arg278 in the case of bIII Similarly, even though IDN5390 interacts with Gln282 in both complexes, it is hydrogen bonded

to Arg278 only in the I3 complex As mentioned above, our results are in accordance with the known pharmacological effects of paclitaxel and of IDN5390 and are able to provide a rational structural basis for them This prompted us to investigate the mode of binding of the much less well characterized epothilone

A No data about the effect of different isotype com-position of tubulin on the activity of this molecule have been reported so far, therefore we used our pro-cedure to investigate the interactions of epithilone A with the bI- and bIII-tubulin isoforms (indicated as E1 and E3, respectively) The average calculated binding energies of complexes sampled during molecular dynamic simulations and subsequently optimized are shown in Table 2 Our data suggest that epothilone A does not preferentially bind to one of the two iso-forms The molecular details of the predicted interac-tions are shown in Fig 7 The position of the ligand in the E1 and E3 structures is not as similar as in the case of paclitaxel and IDN5390 and, especially in E3, substantially differs from the starting complex In E1, the epothilone is located between the M-loop and helix H7, interacting with them essentially through: (i) hydrogen bonds involving C3 and C7 hydroxyl groups and lateral chains of Arg278 and Gln282, respectively; and (ii) p–p interactions between the thiazole ring and His229 In E3, the ligand is farther away from the M-loop and shifted towards helix H1 and the S9–S10 loop with respect to the E1 complex, and therefore is

Table 2 Free energy, enthalpy and entropy of the drug-protein complexes computed at 300 K between epothilone A and isoforms

I (E1) and III (E3) of human b-tubulin.

Complex

DG (kcalÆmol)1)

DH (kcalÆmol)1)

DS (calÆmol)1)

A

B

Fig 6 Solvent accessible surfaces of the bI (A) and bIII (B)

iso-forms in complex with IDN5390.

able to interact with Gly370 (even if this hydrogen

bond is not consistently observed during throughout

the whole simulation) However, epothilone is still

within the van der Waals distance with His229 and the

carbonyl in C5 is engaged in a hydrogen bond with

Arg278 Also in this case, the structure of the M-loop

differs in the two complexes and is stabilized by a

dif-ferent pattern of hydrogen bonds involving its residues

(in particular, residues 277–280) Similarly to the case

of the P3 and I3 complexes, in E3 the rearrangement

of the loop directs the sidechain of Arg278 towards

the binding pocket, allowing Arg278 to maintain

hydrogen bond interactions with the ligand,

notwith-standing the shift in the ligand position with respect to

the E1 complex Due to the described differences

between the two binding modes, the interactions

invol-ving the ligand in E1 and E3 are quite difficult to

com-pare Nevertheless, the analysis of the two complexes

suggests that there should be no significant difference

in binding energies of epothilone for the two isoforms

These observations, together with the data reported in

Table 2, suggest that epothilone A is able to interact with similar affinities with both the bI and bIII iso-forms of tubulin As a consequence, according to our analysis, it should be useful in cases where resistance mediated by overexpression of the bIII-tubulin isotype arises

Discussion

A combination of molecular modeling and molecular dynamics techniques has been applied to investigate the binding modes of three microtubule stabilizing agents, namely paclitaxel, IDN5390 and epothilone A, with isotypes I and III of human b-tubulin Increased expression of bIII isoform in cancer cells has been cor-related with paclitaxel resistance in several studies, whereas recent findings revealed that the activity of IDN5390 is not affected by bIII-tubulin levels To our knowledge, no data about the activity of epothilones

on tumors characterized by different b-tubulin isotype composition have been reported so far Six complexes

of the three ligands under analysis with the human bI and bIII-tubulin were first built and subjected to molecular dynamics The average binding energies for structures sampled during the simulations were calcula-ted after energy optimization Our data rationalize the experimental observations, suggesting a higher affinity

of paclitaxel for the bI than for the bIII isoform and

an opposite behavior for IDN5390 Interestingly, the calculated binding energies of complexes involving epothilone A are very similar for the bI and bIII iso-forms Although docking simulation results have to be taken with caution, especially when based on modeled structures, our results suggest an equally effective interaction of this molecule with microtubules with dif-ferent isoform composition Representative structures

of complexes sampled during the course of molecular dynamic simulations were subsequently analyzed, with the aim of detecting specific interactions responsible for the differences in the calculated binding energies of paclitaxel and IDN5390 with bI or bIII isotypes Such analysis highlighted the crucial role played by the dif-ferent residue present in the 277 position in the two isoforms (serine in bI and alanine in bIII) in determin-ing the different binddetermin-ing affinities of paclitaxel and IDN5390 to the two distinct isoforms In short, such substitution is responsible for a different rearrange-ment of the M-loop, whose final outcome is a more favorable interaction of paclitaxel and IDN5390 with the bI and bIII isotypes, respectively Our study sup-ports the hypothesis that the molecular basis of the different activities of paclitaxel and IDN5390 against microtubules expressing variable levels of bIII isoform

A

B

Fig 7 Epothilone A in complex with (A) bI-tubulin (E1), and (B)

bIII-tubulin (E3) For sake of clarity, nonpolar hydrogen atoms are

omit-ted and hydrogen bond interactions are represenomit-ted by dashed

lines.

lie in the different ligand binding mode of the two

molecules to the bI and bIII isotypes The same

analy-sis, when applied to epothilone A, predicts that its

binding should not be affected by the isoptype

compo-sition of tubulin, suggesting that this molecule can

have a broader efficacy than paclitaxel and IDN5390

and perhaps be less prone to inducing resistance in

tumor cells

Experimental procedures

Comparative modeling

Human tubulin isoform sequences were downloaded from

the Swiss-Prot database (http://www.expasy.org) The

identifiers of human bI- and bIII-tubulins are P07437

(TBB2_human) and Q13509 (TBB3-human), respectively

The multiple sequence alignments were obtained using

clustalw[25]

The comparative modeling protocol consisted of

import-ing the main-chain coordinates of the conserved regions

from the template and positioning the replaced sidechains

in their most commonly observed conformation [26] The

model was optimized using 100 cycles of steepest descent

energy minimization using discover [27]

The PDB identifiers of the three-dimensional structures

used in this work are as follows: 1FFX, 1IA0, 1JFF, 1SA0,

1TUB and 1TVK

Molecular dynamics

Each complex was subjected to 2000 ps of molecular

dynamic simulations with a time step of 1.5 fs The

calcu-lations were performed using macromodel version 7.2

[31] with the AMBER* united atom force field [32]

Sol-vent effects were taken into account by means of the

implicit GB⁄ SA water model [33] A force constant of

23.9 kcalÆmolÆA˚)1 was applied to the protein backbone,

while sidechains and ligands were left free One hundred

frames were sampled at regular time intervals for each

drug–protein complex and subjected to 5000 steps of the

Polak-Ribiere Conjugate Gradient energy minimization

algorithm with the same force field and parameters as

above During these optimizations all constrains were

removed allowing full relaxation of the system internal

degrees of freedom In order to select the most

representa-tive binding modes, a clustering analysis of the optimized

conformational ensemble was performed In details,

con-formations with an internal energy difference lower than

1 kcalÆmol)1 were duplicated if their RMS deviation, after

superposition of the whole coordinate set, was lower than

0.25 A˚ Binding energies and Boltzmann analysis were

car-ried out using the thermodynamic module of the moline

program [28]

Docking experiments Docking simulations of IDN5390 in the paclitaxel (and epothilone A) binding site of b-tubulin were performed using autodock 3.0.5 software [34] Both the modeled protein (hTUB) and the ligand (IDN5390, after building and minimization with macromodel version 7.2 [31]) were imported in autodock Kollman’s united-atoms partial charges and solvent parameters were added to the protein, while Gasteiger atomic charges were calculated for the lig-and Next, a grid including the binding site of interest was defined and several atom probes (corresponding to the atom types of the ligand) were placed at the grid nodes, in order to calculate the interaction energies between the probe and the protein Grid maps were generated for each atom probe The Lamarckian genetic algorithm [34] was employed to explore the orientation⁄ conformation space of the ligand within the binding pocket In Lamarckian genetic algorithm docking, the number of individuals within the population and the number of runs were both set to 200 A maximum number of 2· 106

energy evaluations and 50 000 generations was allowed, while all the remaining parameters were kept to their default values Finally, the conformation with the lowest estimated free energy of binding (and belonging to a well populated cluster) was selected The reliability of the docking protocol was first assessed by simulating the known binding of paclitaxel The protocol described above was able to correctly reproduce the X-ray coordinates of paclitaxel binding conformation (the super-position between the modeled and the experimental struc-ture of the ligand had an RMSD of 0.91) and all the known interactions between the protein and the ligand were reproduced [23]

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Supplementary material

The following supplementary material is available

online:

Table S1 RMSd values (A˚) after optimal superposition

of the backbone atoms of known tubulin structures

Table S2 RMSd values (A˚) after optimal superposition

of the backbone atoms of ligand binding residues

These are defined as the residues within 4 A˚ of any

atom of either paclitaxel in the 1JFF structure or

epo-thilone in 1TVK They are: Glu2 2, Val23, Asp26,

Glu27, Leu217, Gly225, Asp226, His229, Leu230,

Ala233, Ser236, Phe272, Pro274-Arg278, Arg284, Pro360, Arg369-Leu371 (1JFF numbering) The num-ber of superimposed atoms is 176 per pair with the exception of the superpositions involving 1SA0 due to missing residues in this latter structure

Table S3 Interactions between paclitaxel and tubulin observed in the 1JFF entry as reported by Ligplot (http://www.ebi.ac.uk/thornton-srv/databases/cgi-bin/ pdbsum/)

This material is avalilable as part of the online arti-cle from http://www.blackwell-synergy.com