The assembly-inducing laulimalide/peloruside a binding site on tubulin: molecular modeling and biochemical studies with [³H]peloruside A

We used synthetic peloruside A for the commercial preparation of [3H]peloruside A. The radiolabeled compound bound to preformed tubulin polymer in amounts stoichiometric with the polymer's tubulin content, with an apparent K(d) value of 0.35 μM. A less active peloruside A analogue, (11-R)-peloruside A and laulimalide acted as competitive inhibitors of the binding of the [3H]peloruside A, with apparent K(i) values of 9.3 and 0.25 μM, respectively. Paclitaxel, epothilone B, and discodermolide had essentially no ability to inhibit [3H]peloruside A binding, confirming that these compounds bind to a different site on tubulin polymer. We modeled both laulimalide and peloruside A into the binding site on β-tubulin that was identified by Huzil et al. (J. Mol. Biol. 2008, 378, 1016-1030), but our model provides a more reasonable structural basis for the protein-ligand interaction. There is a more complete desolvation of the peloruside A ligand and a greater array of favorable hydrophobic and electrostatic interactions exhibited by peloruside A at its β-tubulin binding site. In addition, the protein architecture in our peloruside A binding model was suitable for binding laulimalide. With the generation of both laulimalide and peloruside A binding models, it was possible to delineate the structural basis for the greater activity of laulimalide relative to peloruside A and to rationalize the known structure-activity relationship data for both compounds.     

Zampanolide, a potent new microtubule-stabilizing agent, covalently reacts with the taxane luminal site in tubulin α,β-heterodimers and microtubules

Zampanolide and its less active analog dactylolide compete with paclitaxel for binding to microtubules and represent a new class of microtubule-stabilizing agent (MSA). Mass spectrometry demonstrated that the mechanism of action of both compounds involved covalent binding to β-tubulin at residues N228 and H229 in the taxane site of the microtubule. Alkylation of N228 and H229 was also detected in α,β-tubulin dimers. However, unlike cyclostreptin, the other known MSA that alkylates β-tubulin, zampanolide was a strong MSA. Modeling the structure of the adducts, using the NMR-derived dactylolide conformation, indicated that the stabilizing activity of zampanolide is likely due to interactions with the M-loop. Our results strongly support the existence of the luminal taxane site of microtubules in tubulin dimers and suggest that microtubule nucleation induction by MSAs may proceed through an allosteric mechanism.     

Systematic profiling of taxol and its analogues (taxalogues) binding to β-tubulin and molecular analysis of their effects on microtubule stabilization

Microtubules are highly dynamic polymers of α/β-tubulin that represent major components of the cytoskeleton and have been established as an attractive druggable target of tumors. Taxol, also known as Paclitaxel, is a microtubule-stabilizing agent that binds stoichiometrically to a specific site in β-tubulin, where is out of but nearby the interacting interface of α/β-tubulin complex, to improve the complex stability through an allosterically regulatory mechanism. In this study, the systematic binding profile of taxol and its 22 structurally diverse, medicinally relevant analogues (termed as taxalogues) to the specific site of β-tubulin as well as their effects on the α/β-tubulin complex stability were created and investigated by using structural modeling, dynamics simulation, and energetics analysis. Two helices H1 and H2 of β-tubulin were identified as key hotspots at the α/β-tubulin interface to mediate the binding event of β-tubulin to α-tubulin. Taxalogue binding can trigger a conformational displacement in the H1 and H2 to elicit the stabilization (or destabilization) of α/β-tubulin. However, strong taxalogue binding potency to β-tubulin does not mean effective α/β-tubulin stabilization; there is only an indirect, moderate correlation between them. Cell viability assay revealed that the taxalogue-induced α/β-tubulin stabilization, but not the taxalogue binding, directly influences the antitumor activity of taxalogues. The activity increases in the order: SB-T-1214 < docetaxel < taxol < larotaxel < cisplatin < cabazitaxel.     

Structural insight into the role of Gln293Met mutation on the Peloruside A/Laulimalide association with αβ-tubulin from molecular dynamics simulations,binding free energy calculations and weak interactions analysis

Peloruside A (PLA) and Laulimalide (LAU) are novel microtubule-stabilizing agents with promising properties against different cancer types. These ligands share a non-taxoid binding site at the outer surface of β-tubulin and promote microtubule stabilization by bridging two adjacent αβ-tubulin dimers from parallel protofilaments. Recent site-directed mutagenesis experiments confirmed the existence of a unique β-tubulin site mutation (Gln293Met) that specifically increased the activity of PLA and caused resistance to LAU, without affecting the stability of microtubules in the absence of the ligands. In this work, fully atomistic molecular dynamics simulations were carried out to examine the PLA and LAU association with native and mutated αβ-tubulin in the search for structural and energetic evidence to explain the role of Gln293Met mutation on determining the activity of these ligands. Our results revealed that Gln293Met mutation induced the loss of relevant LAU–tubulin contacts but exerted negligible changes in the interaction networks responsible for PLA–tubulin association. Binding free energy calculations (MM/GBSA and MM/PBSA), and weak interaction analysis (aNCI) predicted an increased affinity for PLA, and a weakened association for LAU after mutation, thus suggesting that Gln293Met mutation exerts its action by a modulation of drug–tubulin interactions. These results are valuable to increase understanding about PLA and LAU activity and to assist the future design of novel agents targeting the PLA/LAU binding pocket.     

紫杉醚与αβ微管蛋白的分子对接

采用Insight II/Affinity对紫杉醚与αβ微管蛋白进行分子对接, 共得到10个对接构象. 应用密度泛函B3LYP/6-31G 方法计算对接口袋构象的结合能, 筛选出结合能达-190.53 kJ·mol-1的最优对接构象5. 通过构象分析建立紫杉醚与受体结合的作用模型, 结果表明, 在活性口袋的底部紫杉醚与受体间的作用主要是疏水作用, 而在活性口袋的顶部两者间主要是氢键作用. 氢键作用位置可分为A和B两个作用区, 其中A区有3个氢键, 由C13侧链分别与受体的ASP26和ARG369作用形成; B区也有3个氢键, 是由紫杉醚母环上的极性基团分别与受体的THR276、ARG278和GLN282作用产生的. 紫杉醚与αβ微管蛋白间形成的6个氢键可以有效地将紫杉醚固定在活性口袋中.     

Anticancer activity of chamaejasmine: effect on tubulin protein

In this work, the anticancer activity of chamaejasmine was studied by evaluating its in vitro cytotoxicity against several human cancer cell lines (MCF-7, A549, SGC-7901, HCT-8, HO-4980, Hela, HepG2, PC-3, LNCap, Vero and MDCK) using the MTT assay. Results indicated chamaejasmine showed more notable anticancer activity than taxol against PC-3 cells, with IC?? values of 2.28 and 3.98 μM, respectively. Furthermore, Western blot analysis showed that chamaejasmine was able to increase the expression of β-tubulin, but not α-tubulin. In silico simulations indicated that chamaejasmine specifically interacts with the active site which is located at the top of β-tubulin, thanks to the presence of strong hydrophobic effects between the core templates and the hydrophobic surface of the TB active site. The binding energy (E(inter)) was calculated to be -164.77 kcal·mol?1. Results presented here suggest that chamaejasmine possesses anti-cancer properties relating to β-tubulin depolymerization inhibition, and therefore is a potential source of anticancer leads for the pharmaceutical industry.     

A neurosteroid analogue photolabeling reagent labels the colchicine-binding site on tubulin: a mass spectrometric analysis

Previous studies have shown that the neurosteroid analogue, 6-Azi-pregnanolone (6-AziP), photolabels voltage-dependent anion channels and proteins of approximately 55 kDa in rat brain membranes. The present study used two-dimensional electrophoresis and nanoelectrospray ionization ion-trap mass spectrometry (nano-ESI-MS) to identify the 55 kDa proteins (isoelectric point 4.8) as isoforms of β-tubulin. This identification was confirmed by immunoblot and immunoprecipitation of photolabeled protein with anti-β-tubulin antibody and by the demonstration that 6-AziP photolabels purified bovine brain tubulin in a concentration-dependent pattern. To identify the photolabeling sites, purified bovine brain tubulin was photolabeled with 6-AziP, digested with trypsin, and analyzed by matrix-assisted laser desorption/ionization MS (MALDI). A 6-AziP adduct of TAVCDIPPR(m/z?= 1287.77), a β-tubulin specific peptide, was detected by MALDI. High-resolution liquid chromatography-MS/MS analysis identified that 6-AziP was covalently bound to cysteine 354 (Cys-354), previously identified as a colchicine-binding site. 6-AziP photolabeling was inhibited by 2-methoxyestradiol, an endogenous derivative of estradiol thought to bind to the colchicine site. Structural modeling predicted that neurosteroids could dock in this colchicine site at the interface between α- and β-tubulin with the photolabeling group of 6-AziP positioned proximate to Cys-354.     

Molecular insight of isotypes specific β-tubulin interaction of tubulin heterodimer with noscapinoids

Noscapine and its derivatives bind stoichiometrically to tubulin, alter its dynamic instability and thus effectively inhibit the cellular proliferation of a wide variety of cancer cells including many drug-resistant variants. The tubulin molecule is composed of α- and β-tubulin, which exist as various isotypes whose distribution and drug-binding properties are significantly different. Although the noscapinoids bind to a site overlapping with colchicine, their interaction is more biased towards β-tubulin. In fact, their precise interaction and binding affinity with specific isotypes of β-tubulin in the αβ-heterodimer has never been addressed. In this study, the binding affinity of a panel of noscapinoids with each type of tubulin was investigated computationally. We found that the binding score of a specific noscapinoid with each type of tubulin isotype is different. Specifically, amino-noscapine has the highest binding score of ?6.4, ?7.2, ?7.4 and ?7.3 kcal/mol with αβ_I, αβ_II, αβ_III and αβ_IV isotypes, respectively. Similarly 10 showed higher binding affinity of ?6.8 kcal/mol with αβ_V, whereas 8 had the highest binding affinity of ?7.2, ?7.1 and ?7.2 kcal/mol, respectively with αβ_VI, αβ_VII and αβ_VIII isotypes. More importantly, both amino-noscapine and its clinical derivative, bromo-noscapine have the highest binding affinity of ?46.2 and ?38.1 kcal/mol against αβ_III (overexpression of αβ_III has been associated with resistance to a wide range of chemotherapeutic drugs for several human malignancies) as measured using MM-PBSA. Knowledge of the isotype specificity of the noscapinoids may allow for development of novel therapeutic agents based on this class of drugs.     

Discovery of Novel Diarylamide N-Containing Heterocyclic Derivatives as New Tubulin Polymerization Inhibitors with Anti-Cancer Activity

Tubulin has been regarded as an attractive and successful molecular target in cancer therapy and drug discovery. Vicinal diaryl is a simple scaffold found in many colchicine site tubulin inhibitors, which is also an important pharmacophoric point of tubulin binding and anti-cancer activity. As the continuation of our research work on colchicine binding site tubulin inhibitors, we designed and synthesized a series of diarylamide N-containing heterocyclic derivatives by the combination of vicinal diaryl core and N-containing heterocyclic skeletons into one hybrid though proper linkers. Among of these compounds, compound 15b containing a 5-methoxyindole group exhibited the most potent inhibitory activity against the tested three human cancer cell lines (MGC-803, PC-3 and EC-109) with IC₅₀ values of 1.56 μM, 3.56 μM and 14.5 μM, respectively. Besides, the SARs of these compounds were preliminarily studied and summarized. The most active compound 15b produced the inhibition of tubulin polymerization in a dose-dependent manner and caused microtubule network disruption in MGC-803 cells. Therefore, compound 15b was identified as a novel tubulin polymerization inhibitor targeting the colchicine binding site. In addition, the results of molecular docking also suggested compound 15b could tightly bind into the colchicine binding site of β-tubulin.     

Design of a heterobivalent ligand to inhibit IgE clustering on mast cells

We describe the design, synthesis, and characterization of a heterobivalent ligand (HBL) system that competitively inhibits allergen binding to mast cell bound IgE antibody, thereby inhibiting mast cell degranulation. HBLs are composed of a hapten conjugated to a nucleotide analog allowing simultaneous targeting of the antigen-binding site as well the "unconventional nucleotide binding site" on IgE Fab domains. Simultaneous bivalent binding to both sites provides HBLs with over 100-fold enhancement both in avidity for IgE(DNP) (K(d) = 0.33 μM) and in inhibition of allergen binding to IgE(DNP) (IC(50) = 0.45 μM) than the monovalent hapten (K(d)(mono) = 41 μM; IC(50)(mono) = 55.4 μM, respectively). In cellular assays, HBL2 effectively inhibits mast cell degranulation (IC(50) = 15 μM), whereas no inhibition is detected by the monovalent hapten. In conclusion, this study establishes the use of multivalency in a novel HBL design to inhibit mast cell degranulation.     

Exploration of interaction zones of β-tubulin colchicine binding domain of helminths and binding mechanism of anthelmintics

Numerous studies postulated the possible modes of anthelmintic activity by targeting alternate or extended regions of colchicine binding domain of helminth β-tubulin. We present three interaction zones (zones vide −1 to −3) in the colchicine binding domain of Haemonchus contortus (a helminth) β-tubulin homology model and developed zone-wise structure-based pharmacophore models coupled with molecular docking technique to unveil the binding hypotheses. The resulted ten structure-based hypotheses were then refined to essential three point pharmacophore features that captured recurring and crucial non-covalent receptor contacts and proposed three characteristics necessary for optimal zone-2 binding: a conserved pair of H bond acceptor (HBA to form H bond with Asn226 residue) and an aliphatic moiety of molecule separated by 3.75 ± 0.44 Å. Further, an aliphatic or a heterocyclic group distant (11.75 ± 1.14 Å) to the conserved aliphatic site formed the third feature component in the zone-2 specific anthelmintic pharmacophore model. Alternatively, an additional HBA can be substituted as a third component to establish H bonding with Asn204. We discern that selective zone-2 anthelmintics can be designed effectively by closely adapting the pharmacophore feature patterns and its geometrical constraints.     

Synthesis and evaluation of inhibitors of E. coli PgaB, a polysaccharide de-N-acetylase involved in biofilm formation

Many medically important biofilm forming bacteria produce similar polysaccharide intercellular adhesins (PIA) consisting of partially de-N-acetylated β-(1 → 6)-N-acetylglucosamine polymers (dPNAG). In Escherichia coli, de-N-acetylation of the β-(1 → 6)-N-acetylglucosamine polymer (PNAG) is catalysed by the carbohydrate esterase family 4 deacetylase PgaB. The de-N-acetylation of PNAG is essential for productive PNAG-dependent biofilm formation. Here, we describe the development of a fluorogenic assay to monitor PgaB activity in vitro and the synthesis of a series of PgaB inhibitors. The synthesized inhibitors consist of a metal chelating functional group on a glucosamine scaffold to target the active site metal ion of PgaB. Optimal inhibition was observed with N-thioglycolyl amide (K(i) = 480 μM) and N-methyl-N-glycolyl amide (K(i) = 320 μM) glucosamine derivatives. A chemoenzymatic synthesis of an N-thioglycolyl amide PNAG pentasaccharide led to an inhibitor with an improved K(i) of 280 μM.     

Competitive mass spectrometry binding assay for characterization of three binding sites of tubulin

Tubulin is an attractive and established target for anticancer therapy. To date, the only method to determine the binding of inhibitor to tubulin has been competitive radioligand binding assays. We developed a non‐radioactive mass spectrometry (MS) binding assay to study the tubulin binding of colchicine, vinblastine and paclitaxel and to identify which of these three binding sites that a novel inhibitor binds. The method involves a very simple step of separating the unbound ligand from macromolecules using ultrafiltration. The unbound ligand in the filtrate can be accurately determined using highly sensitive and specific liquid chromatography tandem mass spectrometry (LC‐MS/MS) method using multiple reaction monitoring (MRM) mode. The assay was validated using podophyllotoxin, vincristine and docetaxel, drugs that compete to the colchicine‐, vinblastine‐ and paclitaxel‐binding sites in tubulin, respectively. This competitive binding assay allowed the reliable detection of interactions of these drugs with three binding sites on tubulin. This method was subsequently applied to determine the tubulin‐binding site of 4‐substituted methoxylbenzoyl‐aryl‐thiazoles (SMART‐H), a potent antitubulin agent developed in our laboratory. The results indicated that SMART‐H specifically and reversibly bound only to the colchicine‐binding site, but not to vinblastine‐ or paclitaxel sites. This new non‐radioligand binding method to determine the binding site on tubulin will function as a useful tool to study the binding sites of tubulin inhibitors. Copyright © 2010 John Wiley & Sons, Ltd.     

Indole-derived chalcones as anti-dermatophyte agents: In vitro evaluation and in silico study

A series of indole-derived methoxylated chalcones were described as anti-dermatophyte agents. The in vitro antifungal susceptibility testing against different dermatophytes revealed that most of compounds had potent activity against the dermatophyte strains. In particular, the 4-ethoxy derivative 4d with MIC values of 0.25−2 μg/ml was the most potent compound against Trichophyton interdigitale, Trichophyton veruccosum and Microsporum fulvum. Moreover, the 4-butoxy analog 4i displaying MIC values in the range of 1−16 μg/ml had the highest inhibitory activity against Trichophyton mentagrophytes, Microsporum canis, and Arthroderma benhamiae. To predict whether the synthesized compounds interact with tubulin binding site of dermatophytes, the 3D-structure of target protein was modeled by homology modeling and then used for molecular docking and molecular dynamics (MD) simulation studies. Docking simulation revealed that the promising compound 4d can properly bind with tubulin. The molecular dynamics analysis showed that interactions of compound 4d with the active site of target protein have binding stability throughout MD simulation. The results of this study could utilize in the design of more effective antifungal drugs with tubulin inhibition mechanism against keratinophilic fungi.     

Potent taccalonolides, AF and AJ, inform significant structure-activity relationships and tubulin as the binding site of these microtubule stabilizers

The taccalonolides are a class of microtubule stabilizing agents isolated from plants of the genus Tacca. In efforts to define their structure-activity relationships, we isolated five new taccalonolides, AC-AF and H2, from one fraction of an ethanol extract of Tacca plantaginea. The structures were elucidated using a combination of spectroscopic methods, including 1D and 2D NMR and HR-ESI-MS. Taccalonolide AJ, an epoxidation product of taccalonolide B, was generated by semisynthesis. Five of these taccalonolides demonstrated cellular microtubule-stabilizing activities and antiproliferative actions against cancer cells, with taccalonolide AJ exhibiting the highest potency with an IC(50) value of 4.2 nM. The range of potencies of these compounds, from 4.2 nM to >50 μM, for the first time provides the opportunity to identify specific structural moieties crucial for potent biological activities as well as those that impede optimal cellular effects. In mechanistic assays, taccalonolides AF and AJ stimulated the polymerization of purified tubulin, an activity that had not previously been observed for taccalonolides A and B, providing the first evidence that this class of microtubule stabilizers can interact directly with tubulin/microtubules. Taccalonolides AF and AJ were able to enhance tubulin polymerization to the same extent as paclitaxel but exhibited a distinct kinetic profile, suggesting a distinct binding mode or the possibility of a new binding site. The potencies of taccalonolides AF and AJ and their direct interaction with tubulin, together with the previous excellent in vivo antitumor activity of this class, reveal the potential of the taccalonolides as new anticancer agents.     

The anticancer natural product pironetin selectively targets Lys352 of alpha-tubulin

Pironetin is a potent inhibitor of tubulin assembly and arrests cell cycle progression in M phase. Analyses of its structure-activity relationships suggested that pironetin covalently binds tubulin. To determine the binding site of pironetin, we synthesized biotinylated pironetin, which inhibited tubulin assembly both in vitro and in situ. The biotinylated pironetin selectively and covalently bound with tubulin. Partial digestion of biotinylated pironetin-treated tubulin by several proteases revealed that the binding site is the C-terminal portion of alpha-tubulin. By systematic alanine scanning, the pironetin binding site was determined to be Lys352 of alpha-tubulin. Lys352 is located at the entrance of a small pocket of alpha-tubulin, and this pocket faces the beta-tubulin of the next dimer. This is the first compound that covalently binds to the alpha subunit of tubulin and Lys352 of alpha-tubulin and inhibits the interaction of tubulin heterodimers.     

The Crystal Structure of 1, 4, 7, 10- Tetraazacyclododecane Hydrate Triperchlorate

1=mTRODUCTIONMacroazacyclicligandisofwideinterestbecauseofitsstructuralsimilaritywiththeskeletonofPOrphyrinringinsomebibectivesubstancessuchaschlorophyllandhemeetc.tl1.Asametalsimulationenzyme,macroazacyclicligandcoordinatedwithtransitionmetalhasbeeninvestigatedthoroghlyinkinetic,dynamicandstructuralcharactert2.3).Butthere'isnorePOrtaboutthecrystalstructureof1,4,7,1o-tetraaza-cyclododecanehydratetriperchlorate.2EXPERIMENTAL2-lSynthesisoftheTitleComplexTetraazacycldodecanewassynthes…     

Formation of ternary complexes by coordination of (diethylenetriamine)-platinum(II) to N1 or N7 of the adenine moiety of the antiviral nucleotide analogue 9

The synthesis of (Dien)Pt(PMEA-N1), where Dien = diethylenetriamine and PMEA2- = dianion of 9-[2-(phosphonomethoxy)ethyl]adenine, is described. The acidity constants of the threefold protonated H3[(Dien)Pt(PMEA-N1)]3+ complex were determined and in part estimated (UV spectrophotometry and potentiometric pH titration): The release of the proton from the (N7)H+ site in H4[(Dien)Pt(PMEA-N1)]3+ occurs with a rather low pKa (= 0.52+/-0.10). The release of the proton from the -P(O)2(OH) group (pKa = 6.69+/-0.03) in H[(Dien)Pt(PMEA-N1)]+ is only slightly affected by the N1-coordinated (Dien)Pt2+ unit. Comparison with the acidic properties of the H[(Dien)Pt(PMEA-N7)]+ species provides evidence that in the (Dien)Pt(PMEA-N7) complex in aqueous solution an intramolecular, outer-sphere macrochelate is formed through hydrogen bonds between the -PO3(2-) residue of PMEA2- and a PtII-coordinated (Dien)NH2 group; its formation degree amounts to about 40%. The stability constants of the M[(Dien)Pt(PMEA-N1)]2+ complexes with M2+ = Mg2+, Ca2+, Ni2+, Cu2+ and Zn2+ were measured by potentiometric pH titrations in aqueous solution at 25 degrees C and I = 0.1 M (NaNO3). Application of previously determined straight-line plots of log K(M(R-PO3))M versus pK(H(R-PO3)H for simple phosph(on)ate ligands. R-PO3(2-), where R represents a non-inhibiting residue without an affinity for metal ions, proves that the primary binding site of (Dien)Pt(PMEA-N1) is the phosphonate group with all metal ions studied; in fact, Mg2+, Ca2+ and Ni2+ coordinate (within the error limits) only to this site. For the Cu[(Dien)Pt(PMEA-N1)]2+ and Zn[(Dien)Pt(PMEA-N1)]2- systems also the formation of five-membered chelates involving the ether oxygen of the -CH2-O-CH2-PO3(2-) residue could be detected; the formation degrees are about 60% and 30%, respectively. The metal-ion-binding properties of the isomeric (Dien)Pt(PMEA-N7) species studied previously differ in so far that the resulting M[(Dien)Pt(PMEA-N7)]2+ complexes are somewhat less stable, but again Cu2+ and Zn2+ also form with this ligand comparable amounts of the mentioned five-membered chelates. In contrast, both M[(Dien)Pt(PMEA-N1/N7)]2+ complexes differ from the parent M(PMEA) complexes considerably; in the latter instance the formation of the five-membered chelates is of significance for all divalent metal ions studied. The observation that divalent metal-ion binding to the phosphonate group of (Dien)Pt(PMEA-N1) and (Dien)Pt(PMEA-N7) is only moderately inhibited (about 0.2-0.4 log units) by the twofold positively charged (Dien)Pt2+ unit at the adenine residue allows the general conclusion, considering that PMEA is a nucleotide analogue, that this is also true for nucleotides and that consequently participation of, for example, two metal ions in an enzymatic process involving nucleotides is not seriously hampered by charge repulsion.     

Evaluation of the effect of the chiral centers of Taxol on binding to β-tubulin: A docking and molecular dynamics simulation study

Taxol is one of the most important anti-cancer drugs. The interaction between different variants of Taxol, by altering one of its chiral centers at a time, with β-tubulin protein has been investigated. To achieve such goal, docking and molecular dynamics (MD) simulation studies have been performed. In docking studies, the preferred conformers have been selected to further study by MD method based on the binding energies reported by the AutoDock program. The best result of docking study which shows the highest affinity between ligand and protein has been used as the starting point of the MD simulations. All of the complexes have shown acceptable stability during the simulation process, based on the RMSDs of the backbone of the protein structure. Finally, MM-GBSA calculations have been carried out to select the best ligand, considering the binding energy criteria. The results predict that two of the structures have better affinity toward the mentioned protein, in comparison with Taxol. Three of the structures have affinity similar to that of the Taxol toward the β-tubulin.     

The state of the guanosine nucleotide allosterically affects the interfaces of tubulin in protofilament

The dynamics of microtubules is essential for many microtubule-dependent cellular functions such as the mitosis. It has been recognized for a long time that GTP hydrolysis in αβ-tubulin polymers plays a critical role in this dynamics. However, the effects of the changes in the nature of the guanosine nucleotide at the E-site in β-tubulin on microtubule structure and stability are still not well understood. In the present work, we performed all-atom molecular dynamics simulations of a αβα-tubulin heterotrimer harboring a guanosine nucleotide in three different states at the E-site: GTP, GDP-Pi and GDP. We found that changes in the nucleotide state is associated with significant conformational variations at the α-tubulin N- and β-tubulin M-loops which impact the interactions between tubulin protofilaments. The results also show that GTP hydrolysis reduces αβ-tubulin interdimer contacts in favor of intradimer interface. From an atomistic point view, we propose a role for α-tubulin glutamate residue 254 in catalytic magnesium coordination and identified a water molecule in the nucleotide binding pocket which is most probably required for nucleotide hydrolysis. Finally, the results are discussed with reference to the role of taxol in microtubule stability and the recent tubulin-sT2R crystal structures.