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Comparative Study
. 2009 Dec 15;48(49):11664-77.
doi: 10.1021/bi901351q.

Distinct pose of discodermolide in taxol binding pocket drives a complementary mode of microtubule stabilization

Marina Khrapunovich-Baine  1 , Vilas Menon, Pascal Verdier-Pinard, Amos B Smith 3rd, Ruth Hogue Angeletti, Andras Fiser, Susan Band Horwitz, Hui Xiao
Affiliations
Comparative Study

Distinct pose of discodermolide in taxol binding pocket drives a complementary mode of microtubule stabilization

Marina Khrapunovich-Baine et al. Biochemistry. .

Abstract

The microtubule cytoskeleton has proven to be an effective target for cancer therapeutics. One class of drugs, known as microtubule stabilizing agents (MSAs), binds to microtubule polymers and stabilizes them against depolymerization. The prototype of this group of drugs, Taxol, is an effective chemotherapeutic agent used extensively in the treatment of human ovarian, breast, and lung carcinomas. Although electron crystallography and photoaffinity labeling experiments determined that the binding site for Taxol is in a hydrophobic pocket in beta-tubulin, little was known about the effects of this drug on the conformation of the entire microtubule. A recent study from our laboratory utilizing hydrogen-deuterium exchange (HDX) in concert with various mass spectrometry (MS) techniques has provided new information on the structure of microtubules upon Taxol binding. In the current study we apply this technique to determine the binding mode and the conformational effects on chicken erythrocyte tubulin (CET) of another MSA, discodermolide, whose synthetic analogues may have potential use in the clinic. We confirmed that, like Taxol, discodermolide binds to the taxane binding pocket in beta-tubulin. However, as opposed to Taxol, which has major interactions with the M-loop, discodermolide orients itself away from this loop and toward the N-terminal H1-S2 loop. Additionally, discodermolide stabilizes microtubules mainly via its effects on interdimer contacts, specifically on the alpha-tubulin side, and to a lesser extent on interprotofilament contacts between adjacent beta-tubulin subunits. Also, our results indicate complementary stabilizing effects of Taxol and discodermolide on the microtubules, which may explain the synergy observed between the two drugs in vivo.

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Figures

Figure 1
Figure 1
Effects of Taxol and discodermolide on CET assembly (a) and drug displacement assays in CET and BBT (b). (a) In vitro activities of Taxol and discodermolide using a tubulin polymerization assay. Purified chicken tubulin, stored in a GTP-containing buffer, was diluted to 8 μM in 0.5 M MEM buffer containing 3 M glycerol. The mixture was incubated at 37°C until the absorbance at 350 nm reached a plateau, at which time DMSO (control), 8 μM GTP, and 8 μM drug was added sequentially, and the absorbance was followed for 80 min. The inset is an enlargement of the timeframe immediately before, during, and after drug addition. Thick short vertical arrows point toward the time of addition, while thin horizontal arrows indicate the time point immediately after. (b) Displacement analyses were performed as described in Materials and Methods. Microtubules were assembled in the presence of different concentrations of non-radioactive Taxol or discodermolide and GTP before addition of [3H]Taxol. The amount of tritiated drug, relative to control (0 μM unlabeled drug) was determined. Displacement curves for CET are shown as solid lines and those for BBT are dashed.
Figure 2
Figure 2
Global hydrogen-deuterium exchange for chicken α- (a) and β-tubulin (b) in the presence of GMPCPP alone (control), with Taxol, or with discodermolide. Using HPLC coupled to a LTQ mass spectrometer, the molecular weight of tubulin pre-incubated with GMPCPP/DMSO or GMPCPP/drug was measured at five different time points after further incubation at 37°C in deuterated 0.1 M MEM buffer.
Figure 3
Figure 3
Drug-induced alterations in deuteration referenced against GMPCPP-stabilized microtubules for (a) α-tubulin and (b) β-tubulin. Data indicate the mean ± standard deviation of three separate experiments. Differences in deuteration (ΔHDX) are expressed as mass units. For overlapping peptides, unique residues are indicated in parentheses. All peptides with significant alterations in deuterium incorporation (P
Figure 4
Figure 4
Mapping the differences and similarities between the local HDX profiles of Taxol- and discodermolide-bound microtubules (1JFF). The peptides are color-coded as follows: dark blue = discodermolide is more protective; red = Taxol is more protective; yellow = both drugs provide similar protection; pink = discodermolide is deprotective, while Taxol has no effect; light blue = Taxol is deprotective, while discodermolide has no effect; and green = both drugs provide similar deprotection from deuterium incorporation. Secondary structure designations are shown in black and are based on Nogales et al.
Figure 5
Figure 5
Mapping local HDX alterations induced by Taxol and discodermolide on the lateral interprotofilament interface of a previously constructed chicken tubulin model. Parts of the H1-S2 loop and the M-loop involved in lateral contacts are indicated in parentheses. In black are unaffected regions (αH1-S2 and M-loops). The βM-loop, shown in red, is protected from deuteration by Taxol binding, but not discodermolide. The βH1-S2 loop (blue) shows reduced labeling when discodermolide is bound, but not when Taxol is bound.
Figure 6
Figure 6
Mapping the local HDX alterations on the (a) inter- and (b) intradimer interfaces of the tubulin dimer (1JFF). The peptides are colored according to the code in Fig. 4. Briefly, peptic peptides labeled in blue are more protected by discodermolide; in red are more protected by Taxol; in yellow are protected similarly by both drugs; in green are deprotected similarly by both drugs; and in pink are deprotected by discodermolide, but unaffected by Taxol. Secondary structure designations are based on Löwe et al.
Figure 7
Figure 7
Contact maps of Taxol (a) and discodermolide (b) with chicken erythrocyte tubulin. In color (red for Taxol, and blue for discodermolide) are the residues in β-tubulin that make primary contacts with the corresponding drug. Clear shapes represent secondary contacts, those that interact with primary ones, but not the drug itself. Residues in squares are represented in both contact maps, but as primary contacts in one and secondary in the other. Residues in diamonds are unique to the corresponding drug. Those in circles are shared between the two drugs. Plusses and zeros are used to designate correspondence with changes in HDX of peptides containing the indicated amino acid residues: ++ indicates a large significant decrease in labeling (ΔHDX > 0.5), + indicates a small but significant decrease in labeling (ΔHDX>0), and 0 indicates no effect on labeling (ΔHDX=0). In (a), residues that have been previously shown to interact with Taxol are boxed in bold.
Figure 8
Figure 8
Structural representation of the discodermolide and Taxol binding sites. Shown in grey and in the same orientation are cartoon representations of the β-tubulin taxane binding pocket with docked discodermolide (a) and Taxol (b). In (a) all secondary structure designations are labeled in red and are based on Löwe et al.(8) In all parts of the figure (a–c) docked Taxol is shown in red and discodermolide in blue. Amino acids that form direct contacts with both drugs are shown in green (colored circles in Fig. 7). In blue are the amino acids that make unique interactions with discodermolide (blue squares in Fig. 7b). Residues that make unique contacts with Taxol and are part of the M-loop (red diamonds in Fig. 7a) are shown in magenta, while those outside the M-loop (red squares in Fig. 7a) are light pink.
Figure 9
Figure 9
Specific interactions of discodermolide with β-tubulin isolated from chicken erythrocytes. The table lists specific contacts made between the residues in the β-tubulin taxane binding pocket (Figs. 7b and 8c) and the atoms in discodermolide. In parentheses is the number of contacts formed between the specified atom and the corresponding amino acid. In bold are the atoms that form hydrogen bonds with the corresponding amino acids in β-tubulin. The moieties involved in hydrogen bond formation are also bold and underlined in the structural representation of discodermolide. In italics are the discodermolide atoms that make either van der Waals or polar contacts with the corresponding residues in β-tubulin. The rest are hydrophobic interactions.
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References

    1. Rowinsky EK. The development and clinical utility of the taxane class of antimicrotubule chemotherapy agents. Annu Rev Med. 1997;48:353–374. - PubMed
    1. Rao S, Horwitz SB, Ringel I. Direct photoaffinity labeling of tubulin with taxol. J Natl Cancer Inst. 1992;84:785–788. - PubMed
    1. Schiff PB, Fant J, Horwitz SB. Promotion of microtubule assembly in vitro by taxol. Nature. 1979;277:665–667. - PubMed
    1. Rao S, He L, Chakravarty S, Ojima I, Orr GA, Horwitz SB. Characterization of the Taxol binding site on the microtubule. Identification of Arg(282) in beta-tubulin as the site of photoincorporation of a 7-benzophenone analogue of Taxol. J Biol Chem. 1999;274:37990–37994. - PubMed
    1. Rao S, Krauss NE, Heerding JM, Swindell CS, Ringel I, Orr GA, Horwitz SB. 3′-(p-azidobenzamido)taxol photolabels the N-terminal 31 amino acids of beta-tubulin. J Biol Chem. 1994;269:3132–3134. - PubMed

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