Decision letter: Structural insight into the stabilization of microtubules by taxanes

Abstract

Article Figures and data Abstract Editor's evaluation Introduction Results Discussion Materials and methods Data availability References Decision letter Author response Article and author information Metrics Abstract Paclitaxel (Taxol) is a taxane and a chemotherapeutic drug that stabilizes microtubules. While the interaction of paclitaxel with microtubules is well described, the lack of high-resolution structural information on a tubulin-taxane complex precludes a comprehensive description of the binding determinants that affect its mechanism of action. Here, we solved the crystal structure of baccatin III the core moiety of paclitaxel-tubulin complex at 1.9 Å resolution. Based on this information, we engineered taxanes with modified C13 side chains, solved their crystal structures in complex with tubulin, and analyzed their effects on microtubules (X-ray fiber diffraction), along with those of paclitaxel, docetaxel, and baccatin III. Further comparison of high-resolution structures and microtubules’ diffractions with the apo forms and molecular dynamics approaches allowed us to understand the consequences of taxane binding to tubulin in solution and under assembled conditions. The results sheds light on three main mechanistic questions: (1) taxanes bind better to microtubules than to tubulin because tubulin assembly is linked to a βM-loopconformational reorganization (otherwise occludes the access to the taxane site) and, bulky C13 side chains preferentially recognize the assembled conformational state; (2) the occupancy of the taxane site has no influence on the straightness of tubulin protofilaments and; (3) longitudinal expansion of the microtubule lattices arises from the accommodation of the taxane core within the site, a process that is no related to the microtubule stabilization (baccatin III is biochemically inactive). In conclusion, our combined experimental and computational approach allowed us to describe the tubulin-taxane interaction in atomic detail and assess the structural determinants for binding. Editor's evaluation Here Prota et al. compare the action of the microtubule-stabilizing agent, taxol, with that of closely-related analogues and, as a result, successfully dissect the interactions and roles of different regions of the taxol molecule. The overall story is solid, providing new molecular insights, including defining and separating the lattice expansion effect from the lattice stabilization effect upon taxane binding. This important work will be of interest to the microtubule cytoskeleton and structural biology communities. https://doi.org/10.7554/eLife.84791.sa0 Decision letter eLife's review process Introduction The taxane paclitaxel is a drug included in the World Health Organization’s List of Essential Medicines (World Health Organization, 2021). Taxanes, either alone or in combination with other chemotherapeutic agents, are important drugs for the treatment of several solid tumors, such as ovarian, lung, and breast cancer, as well as advanced Kaposi’s sarcoma (Ettinger, 1993; Arbuck et al., 1994; Saville et al., 1995; Lindemann et al., 2012). The three taxanes in clinical use, paclitaxel (Taxol), docetaxel (Taxotere), and cabazitaxel (Jevtana), are part of a large family of chemically diverse compounds that bind to the so-called ‘taxane site’ of the αβ-tubulin heterodimer (Field et al., 2013; Steinmetz and Prota, 2018; Figure 1A and B), the building block of microtubules. However, the appearance of peripheral sensory neuropathy and other side effects caused by taxanes compromises treatment efficacy in the long term (Gornstein and Schwarz, 2014). Thus, understanding the underlying mechanism of microtubule stabilization by this class of antitubulin agents is an important requirement for future and safer drug development efforts. Figure 1 Download asset Open asset Structures of tubulin and ligands employed in the work. (A) Tubulin heterodimer (α-tubulin in gray and β-tubulin in white) in ribbon representation, where nucleotide binding sites have been highlighted in sphere representation (B) Structural features of the tubulin β-subunit. (C) Structures of taxanes used in this study. Because taxane-site ligands stabilize microtubules and suppress their dynamics, they are collectively called microtubule-stabilizing agents. Several structures of microtubules in complex with taxane-site agents have been recently analyzed and solved by cryo-electron microscopy (cryo-EM) to resolutions ranging between ~3 and ~10 Å. For paclitaxel, it was initially suggested that the drug acts on longitudinal tubulin contacts along protofilaments in microtubules by allosterically expanding the microtubule lattice in the direction of its long filament axis (Vale et al., 1994; Arnal and Wade, 1995; Alushin et al., 2014), a notion that is also consistent with X-ray fiber diffraction data (Estévez-Gallego et al., 2020). However, more recent studies suggest that paclitaxel enhances lattice flexibility and acts on lateral tubulin contacts between protofilaments in microtubules through interactions with the M-loop of the β-tubulin subunit (βM loop) (Kellogg et al., 2017; Debs et al., 2020; Manka and Moores, 2018). Besides directly acting on microtubules, taxane-site ligands also have the capacity to bind to unassembled tubulin dimers and promote their assembly into microtubules (Schiff and Horwitz, 1981; Carlier and Pantaloni, 1983; Howard and Timasheff, 1988; Díaz et al., 1993; Buey et al., 2004). Several structures of non-taxane agents bound to the taxane site of tubulin have been solved to resolutions ranging from 2.4 to 1.8Å by X-ray crystallography (Prota et al., 2013a; Trigili et al., 2016; Prota et al., 2017; Balaguer et al., 2019). These data suggested that one mode of action of some taxane-site ligands such as zampanolide (PDB ID 4I4T) or epothilone A (PDB ID 4I5O) on unassembled tubulin is to stabilize lateral tubulin contacts between protofilaments within microtubules by structuring and stabilizing the βM loop into a short α-helix (Prota et al., 2013a). In contrast, the absence of a helical structure for this segment in the presence of the taxane-site ligands dictyostatin (PDB ID 4MF4) and discodermolide (PDB ID 5LXT) (Trigili et al., 2016; Prota et al., 2017) suggests a different, still poorly understood mechanism of microtubule stabilization for these two classes of non-taxane agents. In the case of taxanes, one hypothesis is that they preferentially bind to a specific conformation of tubulin. It is well established that tubulin displays two prominent conformations that are related to its assembly state (reviewed in Knossow et al., 2020): a ‘straight’ conformation present in assembled microtubules (denoted ‘straight tubulin’ hereafter) and a ‘curved’ conformation observed in unassembled tubulin (denoted ‘curved tubulin’ hereafter). The ‘curved-to-straight’ conformational transition is required for the formation of lateral tubulin contacts between protofilaments in the main shaft of microtubules. Some data suggest that the activation mechanism of taxanes facilitates the curved-to-straight conformational transition by preferentially binding to the straight conformation of tubulin (Nogales et al., 1998; Elie-Caille et al., 2007; Benoit et al., 2018). Structural information of a taxane in complex with unassembled tubulin is currently unavailable. With the aim of providing insight into the mechanism of action of this important class of anticancer drugs and into the tubulin-taxane interaction, we solved the high-resolution structures of three different taxanes bound to curved tubulin by X-ray crystallography. We further analyzed the effects of different taxanes on the microtubule lattice by X-ray fiber diffraction. These studies were complemented with molecular dynamics (MD) simulations that shed light on issues that were not amenable to experimental verification. Taken together, our results suggest that the main reason for the differential affinity of taxane-site ligands for assembled tubulin and unassembled tubulin arises from two terms. First, the stabilization of the βM loop in an ‘out’ conformation compatible with the formation of specific lateral contacts in microtubules and second, the selectivity of the bulky C13 side chain for the assembled, straight conformational state of tubulin. Finally, we found that the occupancy of the taxane site results in a displacement of the S9-S10 loop of β-tubulin that accounts for the observed microtubule expansion with no influence, however, on the straightness of tubulin protofilaments. Results High-resolution crystal structure of a tubulin-taxane complex To determine the high-resolution structure of a taxane bound to curved tubulin, we performed both soaking and co-crystallization experiments using the previously described protein complexes termed T2R-TTL and TD1. The former complex is composed of two αβ-tubulin heterodimers bound head-to-tail, the stathmin-like protein RB3, and the tubulin tyrosine ligase (PDB ID 4IIJ) (Prota et al., 2013a; Prota et al., 2013b); the latter complex contains one αβ-tubulin heterodimer and the DARP in D1 (PDB ID 4DRX) (Pecqueur et al., 2012). We did not succeed in procuring any valuable structural information from these two crystal ensembles using a first series of taxanes comprising paclitaxel, docetaxel, the more soluble 3’-N-m-aminobenzamido-3’-N-debenzamidopaclitaxel (N-AB-PT) (Li et al., 2000), and the engineered, high-affinity taxanes Chitax 40 (Matesanz et al., 2008) and Chitax 68 (Ma et al., 2018). We thus decided to approach the issue from a different angle and started off with baccatin III, a precursor in the biosynthesis of paclitaxel that contains both the C2-benzoyloxy ring C and the C10 acetate ester, but lacks the C13 side chain with both the 3’-N-benzamido phenyl ring A and the 3’-phenyl ring B moieties (Samaranayake et al., 1993; Figure 1C). Notably, baccatin III is largely biologically inactive despite displaying micromolar affinity for microtubules (Parness et al., 1982; Lataste et al., 1984; Kingston, 2000; Andreu and Barasoain, 2001). We found that baccatin III shows detectable affinity (Kb 25°C 3.0±0.5 × 103 M–1) to unassembled tubulin, which is in the same range as for other compounds that have been co-crystallized with tubulin, such as epothilone A 8±3 × 103 M–1 (Canales et al., 2014) and discodermolide 2.0±0.7 × 104 M–1 (Canales et al., 2011). Therefore, we hypothesized that the presence of the C13 side chain of the aforementioned taxanes might preclude the binding to the curved tubulin form present in both the T2R-TTL and the TD1 complexes. Subsequently, we succeeded in obtaining a T2R-TTL-baccatin III complex structure that was solved at 1.9 Å resolution (PDB ID 8BDE) (Figure 2A and D; Table 1). We found that the ligand binds to the taxane site of curved tubulin with its C2-benzoyloxy ring C stacked between the side chains of βH229 and βL275 in the leucine-rich β-tubulin pocket lined by the side chains of βC213, βL217, βL219, βD226, βH229, βL230, and βL275 (Figures 3A and 4A). Its carbonyl oxygen forms a weak hydrogen bond to the main chain amide of βR278. The C10 acetate is exposed to the solvent and, together with the C12 methyl, is within van der Waals distance to βG370 of the βS9-βS10 loop. Furthermore, the oxetane oxygen and the C13 hydroxyl accept hydrogen bonds from the main chain amide nitrogen of βT276 and the βH229 imidazole NE2, respectively. The C4 acetate is buried in the hydrophobic pocket made up by βL230, βA233, βF272, βP274, βL275, βM302, βL371, and the aliphatic portion of the βR369 side chain. Figure 2 Download asset Open asset T2R-TTL structures in complex with baccatin III, 2a, and 2b. Overall view of the T2R-TTL-baccatin III (PDB ID 8BDE) (A), the T2R-TTL-2a (PDB ID 8BDF) (B), and the T2R-TTL-2b (PDB ID 8BDG) crystal structures. The α- and β-tubulin chains are colored in dark and light gray, respectively. The TTL chains (cyan) and the RB3 (yellow-orange) are shown in ribbon representation. The tubulin-bound ligands are displayed as spheres and are colored following the same color scheme as in the main figures. (D–F) Electron-density maps highlighting the bound baccatin III, 2a, and 2b. The SigmaA-weighted 2mFo − DFc (dark blue mesh) and mFo − DFc (light green mesh) omit maps are contoured at +1.0σ and +3.0σ, respectively. The map calculations excluded the atoms of the corresponding ligands. (G) Anomalous density peaks detected in both the binding sites in chains B and D of T2R-TTL for the bromine moiety of compound 2b. Figure 3 Download asset Open asset Crystal structure of T2R-TTL-baccatin III (PDB ID 8BDE) and T2R-TTL-2a (PDB ID 8BDF) complexes. (A) Close-up view of the interaction network observed between baccatin III (lemon) and β-tubulin (light gray). Interacting residues of tubulin are shown in stick representation and are labeled. Oxygen and nitrogen atoms are colored red and blue, respectively; carbon atoms are in lemon (baccatin III) or light gray (tubulin). Hydrogen bonds are depicted as black dashed lines. Secondary structural elements of tubulin are labeled in blue. (B) Close-up view of the interaction of 2a (violet) with β-tubulin in the same view and representation as in (A). (C) The same close-up view as in (A) and (B) with the superimposed baccatin III (lemon) and 2a (violet) complex structures. Water molecules belonging to the baccatin III structure are represented as lemon spheres. Figure 4 Download asset Open asset Comparison of taxane binding to unassembled curved versus assembled straight tubulin. (A) Close-up view of the superimposed baccatin III bound (ligand in lemon; protein in gray ribbon and sticks) to curved tubulin (PDB ID 8BDE) and paclitaxel bound to straight tubulin as found in a microtubule (PDB ID 6WVR; ligand in dark green; protein in slate ribbon and sticks) structures. Interacting residues of tubulin are shown in stick representation and are labeled. Oxygen and nitrogen atoms are colored red and blue, respectively. Hydrogen bonds are depicted as black dashed lines. Secondary structural elements of tubulin are labeled in blue. Water molecules belonging to the baccatin III structure are represented as lemon spheres. The structures were superimposed onto their taxane sites (residues 208–219+225–237+272–276+286–296+318–320+359–376); root-mean-square deviations (rmsd) 0.894 Å (52 Cα atoms). (B) Close-up view of superimposed 2a bound to curved tubulin (PDB ID 8BDF) (ligand in violet; protein in gray ribbon and sticks) and paclitaxel bound to straight tubulin (PDB ID 6WVR; ligand in dark green; protein in slate ribbon and sticks) structures (rmsd 0.826 Å over 52 Cα atoms) using the same settings as in (A). (C) Conformational changes on β-tubulin induced by paclitaxel upon binding to straight tubulin in microtubules (PDB ID 6WVR). The α-tubulin and β-tubulin chains are in ribbon representation and are colored in dark and light gray, respectively. The rmsd differences between unbound and paclitaxel-bound straight tubulin are represented as dark (backbone rmsd) blue spheres. Only the rmsd differences above a threshold of average ± standard deviation are displayed. The sphere radii correspond to the average-subtracted rmsd values displayed in panel (D). (D) Rmsd plots of backbone positions between the paclitaxel bound (PDB ID 6WVR) and the apo (PDB ID 6DPV) straight tubulin in microtubules. The gray error bar represents the average rmsd ± standard deviation. The top bar is colored according to the following domain assignment: N-terminal domain (N-domain., marine blue), intermediate domain (I-domain, orange), central helix βH7 (lemon), and C-terminal domain (C-domain, red). The β-tubulin chains of the corresponding structures were superimposed onto their β-tubulin N-terminal β-sheets (rmsd 0.304 Å over 30 Cα). Table 1 X-ray data collection and refinement statistics. T2R-TTL-BacIIIT2R-TTL-2aT2R-TTL-2bData collectionSpace groupP212121P212121P212121Cell dimensionsa, b, c (Å)104.1, 157.2, 179.2104.8, 157.9, 179.1105.3, 158.6, 179.2Resolution (Å)49.2–1.9 (1.95–1.90)49.3–1.95 (2.00–1.95)49.4–2.35 (2.41–2.35)Rmerge(%)10.7 (491.9)13.3 (516.6)17.4 (403.5)Rmeas (%)11.1 (513.1)13.6 (526.1)17.7 (410.8)Rpim (%)3.3 (147.5)2.9 (102.9)2.6 (57.7)I/σI16.5 (0.5)20.1 (0.7)20.1 (0.9)CC half100 (17.8)100 (31.4)99.9 (46.6)Completeness (%)100 (99.8)100 (100)100 (100)Redundancy13.5 (12.4)27.3 (27.8)28.5 (28.3)RefinementResolution (Å)49.2–1.949.3–1.9549.4–2.35No. unique reflections229654215774125168Rwork/Rfree19.2/21.818.9/21.618.3/21.4No. atomsProtein175551738917227Ligand42120Water861883166Average B-factors (Å2)Protein59.062.976.1Ligand (chain B/D)n.a. / 109.2111.4/102.8146.6/144.9Water56.260.359.4Wilson B-factor41.743.156.9R.m.s. deviationsBond lengths (Å)0.0030.0030.002Bond angles (°)0.6420.6550.550Ramachandran statisticsFavored regions (%)98.198.198.0Allowed regions (%)1.81.82.0Outliers (%)0.10.10 For each structure, data were collected from a single crystal. Values in parentheses are for highest-resolution shell. Generation of paclitaxel analogs that bind to tubulin crystals Aiming to understand the implication on tubulin activation of the paclitaxel’s bulky and hydrophobic C13 ring A moiety (or its equivalent tert-butyl in docetaxel) and to elucidate the reason why it apparently precludes binding to T2R-TTL and TD1 crystals (see above), we devoted a synthetic effort to obtaining new taxane ligands with modified C13 side chains. We produced a series of modified taxanes bearing smaller groups than paclitaxel at the 3’-N position, namely, acrylamide 2a, haloacetamides 2b, and 2c, and isothiocyanate 2d (Figure 1C). We could measure binding of 2a to unassembled tubulin dimers (Kb25°C 0.8±0.3 × 103 M–1), but not of N-AB-PT (Li et al., 2000), Chitax 40 (Matesanz et al., 2008), or Chitax 68 (Ma et al., 2018), thus indicating that the modification of the paclitaxel structure increased the binding affinity for unassembled tubulin. In fact (Figure 2B, C, E and F), we found unequivocal difference electron densities at the taxane site of β-tubulin in T2R-TTL crystals soaked with 2a (PDB ID 8BDF) and 2b (PDB ID 8BDG) and refined the corresponding structures to 1.95 and 2.35 Å resolution, respectively (Table 1). Interestingly, the electron densities of compounds 2a and 2b displayed a continuity between the 3’-N-attached moieties of both ligands and the side chain of His 229 of β-tubulin (βH229), suggesting the possible formation of a covalent adduct. For further validation, we collected additional X-ray diffraction data on T2R-TTL crystals soaked with the haloacetamide derivative 2b at the bromine peak wavelength of 0.91501 Å. After rigid body and restrained refinement, we detected two clear anomalous difference peaks in electron densities at the taxane sites of the two tubulin dimers in the T2R-TTL crystals soaked with 2b (Figure 2G), which did not support covalent bond formation. Furthermore, refinement cycles performed in parallel with 2a modeled in both the covalent and the non-covalent form, resulted in clear electron density for the non-covalent model, while red difference peaks for the covalent form were always present after refinement (not shown). Accordingly, we interpreted the continuous electron density observed in the T2R-TTL-2a structure as a strong hydrogen bond between the βH229 NE2 and the C39 carbonyl of the ligand side chain rather than a covalent bond (Figure 3B). The T2R-TTL-2a complex structure revealed that 2a engages in comparable interactions to curved tubulin by means of both its C2-benzoyloxy ring C and its oxetane moieties, as found for baccatin III (Figure 3A and B). However, the core ring system of 2a is tilted toward helix βH6 and strand βS7 by ~20° (angle between the two C1-C9 axes; rmsdbacIII-2a of 0.794 Å for 39 core atoms), thereby adopting a pose that is closer to that observed for paclitaxel bound to straight tubulin in microtubules (PDB ID 6WVR; rmsd2a-paclitaxel of 0.845 Å for 56 core atoms; rmsdbacIII-paclitaxel of 1.048 Å for 42 core atoms; Figure 4B). Similar to paclitaxel bound to straight tubulin, the C39 carbonyl of the C13-3’-N-acrylamide moiety of 2a forms a hydrogen bond to the βH229 NE2 in curved tubulin (Figures 3B and 4B). The terminal ester moiety of 2a is exposed to the solvent and it forms water-mediated hydrogen bonds to the side chains of βE22 and βR369 of β-tubulin; it lodges within a space that is otherwise occupied by crystallographic water molecules in the curved tubulin-baccatin III structure. This favorable hydrogen bond network cannot be established by the 3’-N-benzamido phenyl ring A of paclitaxel in the curved tubulin conformation. Moreover, both the water molecules and the hydrophobic portions of the Lys19, Glu22, and Val23 side chains on helix H1 are too far apart for being able to provide favorable forces to stabilize the ring A. In the context of paclitaxel-bound microtubules (straight tubulin), the helix H1 moves closer toward helix H7, thereby allowing these three side chains to form a hydrophobic cavity that stabilizes the A ring, which suggest a structural mechanism for the higher affinity of paclitaxel observed for the straight tubulin conformation. Moreover, the helix H1 movement causes the side chain of βD26 to occupy the space of the βR369 side chain, which adopts a flipped-out conformation. This arrangement provides additional stabilization through a polar interaction to the 3’ amide nitrogen of paclitaxel and supports a more favorable binding of paclitaxel to microtubules (Figures 3B and 4B). The absence of the C10 acetate in 2a relative to baccatin III has little impact on the conformation of the secondary structural elements that shape the taxane site (Figure 3C). Together, these structural data provide – for the first time – a high-resolution structural description of the interaction of taxanes harboring a C13 side chain with unassembled, curved tubulin. They indicate that the main interaction energy of this class of antitubulin agents is mediated by their common baccatin III core moieties. They further reveal that the taxane pose in both curved and straight tubulin is very similar; however, subtle structural details reveal why paclitaxel binds more favorably to straight tubulin. The knowledge of these structural determinants may support the development of next-generation taxanes to better tune their mechanism of action, thereby opening a new window to control undesired side effects. Overall, our results suggest that the tubulin-2a structure is an excellent model to study the interaction of paclitaxel with curved tubulin at high resolution and that X-ray crystallography is a valuable method to analyze the molecular mechanism of action of microtubule-stabilizing agents binding to the taxane site. Conformational changes upon taxane binding to curved and straight tubulin Next, we investigated the conformational changes induced by binding of baccatin III and 2a to curved tubulin. To this end, we first superimposed the crystal structures of apo tubulin (PDB ID 4I55), tubulin-baccatin III (PDB ID 8BDE), and tubulin-2a (PDB ID 8BDF) onto the N-terminal β-sheets of β-tubulin (residues 3–9, 63–66, 132–138, 163–169, and 198–202), and calculated the root-mean-square deviations (rmsd) between the apo and the two ligand-bound states (rmsdBacIII 0.08 Å of 29 Cα; rmsd2a 0.10 Å of 29 Cα). These rmsd values were also plotted and mapped onto the corresponding structures to highlight the major regions of conformational change. As shown in Figure 5, significant and comparable conformational changes were observed for backbone atoms of the βT5 loop and the N-terminal segment of the βM loop in both the tubulin-baccatin III and tubulin-2a complex structures. Interestingly, the βT5 loop that is prominently involved in establishing longitudinal tubulin contacts along protofilaments is oriented in the active ‘out’ conformation in both structures (Nawrotek et al., 2011). This observation indicates an allosteric crosstalk between the taxane site and the βT5 loop possibly via the central helix βH7 and the guanosine nucleotide bound to β-tubulin. In the case of the βM loop, we only found well-defined electron densities for its N-terminal section up to residue βR278, while the remaining portion of the loop appeared disordered in both complex structures. This partial βM loop structuring has been observed previously in tubulin complexes with the taxane-site ligands dictyostatin and discodermolide (Trigili et al., 2016; Prota et al., 2017; note that the taxane-site ligands zampanolide and epothilone A promote the structuring of the βM loop into a helical conformation Prota et al., 2013a). A direct effect of taxanes on the βM loop is consistent with the notion that paclitaxel stabilizes this secondary structural element in two discrete conformations giving rise to heterogeneous lateral microtubule-lattice contacts (Debs et al., 2020). We also found significant conformational changes in the βS9-βS10 loop, which were more prominent in tubulin-2a than in tubulin-baccatin III. This finding can be explained by the presence of a C13 side chain in 2a that needs more room for accommodation inside the taxane site compared to baccatin III, which lacks a C13 side chain. Finally, we observed a conformational change of the H2’ helix in the tubulin-baccatin III structure, which was absent in tubulin-2a. Figure 5 Download asset Open asset Conformational changes induced by taxane binding to unassembled, curved tubulin. (A) Conformational changes on the backbone atoms (dark blue) of the β-tubulin chain induced by baccatin III upon binding to curved tubulin. The tubulin chains are in ribbon representation and are colored in dark (α-tubulin) and light (β-tubulin) gray, respectively. The root-mean-square deviation (rmsd) values of the superimposed unbound and baccatin III-bound curved tubulin are represented as dark blue (backbone rmsd) spheres, respectively. Only the rmsd values above a threshold of average + standard deviation are displayed. The sphere radii correspond to the average-subtracted rmsd values displayed in panel (B). (B) Rmsd plots of the backbone (bottom) positions between the baccatin bound (PDB ID 8BDE) and the apo (PDB ID 4I55) curved tubulin state. The gray error bar represents the average rmsd ± standard deviation. The top bar is colored according to the following domain assignment: N-terminal domain (N-domain, marine blue), intermediate domain (I-domain, orange), central helix H7 (lemon), C-terminal domain (C-domain, red). The β-tubulin chains of the corresponding structures were superimposed onto their β-tubulin N-terminal β-sheet (rmsd 0.08 Å over 29 Cα). (C) Conformational changes on the backbone atoms (dark blue) of the β-tubulin chain induced by 2a upon binding to curved tubulin. (D) Rmsd plots of the backbone (bottom) positions between the 2a bound (PDB ID 8BDF) and the apo (PDB ID 4I55) curved tubulin state (rmsd 0.10 Å over 29 Cα). The same display settings as in (B) are applied. To investigate the effect of the observed conformational changes on the relative domain arrangements in β-tubulin of the individual complexes, we further superimposed the β-tubulin chains of apo tubulin, tubulin-baccatin III, and tubulin-2a onto their central βH7 helices (residues 224–243). For tubulin-baccatin III, a subtle relative twist between the N-terminal and the intermediate domains was observed (Figure 6; Videos 1 and 2), while binding of 2a rather caused both the N-terminal and intermediate domains of β-tubulin to move slightly apart (Figure 6; Videos 3 and 4). Thus, taxane binding to tubulin causes global, but subtle conformational rearrangements. Figure 6 Download asset Open asset Schematic representation of subtle domain movements observed from apo to baccatin III- to 2a-bound curved tubulin. The three structures were superimposed onto their central helices βH7 to highlight better the subtle domain movements relative to each other. The individual domains are colored according to their domain assignment and their borders are contoured using the same color scheme: N-terminal domain (N-domain, marine blue), intermediate domain (I-domain, orange), central helix βH7 (lemon), C-terminal domain (C-domain, red). The directions of the individual movements are highlighted with black arrows. Video 1 Download asset This video cannot be played in place because your browser does support HTML5 video. You may still download the video for offline viewing. Download as MPEG-4 Download as WebM Download as Ogg Conformational transition from apo to baccatin III-bound, unassembled tubulin state. Top view on β-tubulin (onto the ‘plus end’ in the context of a microtubule). Video 2 Download asset This video cannot be played in place because your browser does support HTML5 video. You may still download the video for offline viewing. Download as MPEG-4 Download as WebM Download as Ogg Conformational transition from apo to baccatin III-bound, unassembled tubulin state. Luminal view on β-tubulin (view from the lumen in the context of a microtubule). Video 3 Download asset This video cannot be played in place because your browser does support HTML5 video. You may still download the video for offline viewing. Download as MPEG-4 Download as WebM Download as Ogg Conformational transition from apo to 2a-bound, unassembled tubulin state. Top view on β-tubulin (onto the ‘plus end’ in the context of a microtubule). Video 4 Download asset This video cannot be played in place because your browser does support HTML5 video. You may still download the video for offline viewing. Downlo