Macromolecular Accessibility of Fluorescent Taxoids Bound at a Paclitaxel Binding Site in the Microtubule Surface

Díaz, J. Fernando; Barasoaín, Isabel; Souto, André A.; Amat‐Guerri, F.; Andreu, José Manuel

doi:10.1074/jbc.m407816200

Cited by 47 publications

(38 citation statements)

References 53 publications

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“…FRETefficiencies from TAMRA-X-taxol to positions on the kinesin head are largely consistent with cryoelectron microscopy structures that show the orientation of bound kinesin heads and that locate bound taxol at or near the luminal surface of the microtubule. The location of the TAMRA-X-taxol is consistent, within experimental uncertainty, with the position of other fluorescent taxol derivatives determined previously (31).…”

Section: Discussionsupporting

confidence: 67%

FRET measurements of kinesin neck orientation reveal a structural basis for processivity and asymmetry

Martin

Fathi²,

Mitchison

et al. 2010

Proc. Natl. Acad. Sci. U.S.A.

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As the smallest and simplest motor enzymes, kinesins have served as the prototype for understanding the relationship between protein structure and mechanochemical function of enzymes in this class. Conventional kinesin (kinesin-1) is a motor enzyme that transports cargo toward the plus end of microtubules by a processive, asymmetric hand-over-hand mechanism. The coiled-coil neck domain, which connects the two kinesin motor domains, contributes to kinesin processivity (the ability to take many steps in a row) and is proposed to be a key determinant of the asymmetry in the kinesin mechanism. While previous studies have defined the orientation and position of microtubule-bound kinesin motor domains, the disposition of the neck coiled-coil remains uncertain. We determined the neck coiled-coil orientation using a multidonor fluorescence resonance energy transfer (FRET) technique to measure distances between microtubules and bound kinesin molecules. Microtubules were labeled with a new fluorescent taxol donor, TAMRA-X-taxol, and kinesin derivatives with an acceptor fluorophore attached at positions on the motor and neck coiled-coil domains were used to reconstruct the positions and orientations of the domains. FRET measurements to positions on the motor domain were largely consistent with the domain orientation determined in previous studies, validating the technique. Measurements to positions on the neck coiled-coil were inconsistent with a radial orientation and instead demonstrated that the neck coiled-coil is parallel to the microtubule surface. The measured orientation provides a structural explanation for how neck surface residues enhance processivity and suggests a simple hypothesis for the origin of kinesin step asymmetry and "limping."is a dimeric motor enzyme involved in microtubule-based intracellular transport (1). Kinesin utilizes ATP hydrolysis to move toward the plus ends of microtubules via a hand-over-hand mechanism in which each motor domain (or "head") alternately steps forward (2, 3). The mechanism is asymmetrical, in the sense that alternate steps are different structurally and kinetically (2, 4-7), and is processive, in that kinesin can take hundreds of steps along a microtubule before dissociating (8). Both of these mechanistic features may be important to biological function: Asymmetry allows kinesin to use energy for cargo transport rather than dissipating it in cargo rotation; processivity allows individual kinesin molecules to transport cargoes through the viscoelastic cytoplasm.Processive movement requires that kinesin maintain a tight grip on the microtubule as it moves. To this end, the catalytic cycle is gated to ensure that at least one head remains tightly bound to the microtubule at all times (9-11). However, the kinesin-1 neck, a five heptad repeat coiled-coil connected to the two heads by the short nonhelical neck linkers (Fig. 1), also appears to play a role in keeping the enzyme bound to the microtubule: Mutations on the neck surface can significantly increase or decrease processi...

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Section: Discussionsupporting

confidence: 67%

FRET measurements of kinesin neck orientation reveal a structural basis for processivity and asymmetry

Martin

Fathi²,

Mitchison

et al. 2010

Proc. Natl. Acad. Sci. U.S.A.

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“…To further examine the effect of HDAC6 function and tubulin acetylation on paclitaxel binding, we did a series of in vitro paclitaxel-binding experiments, using the fluorescent paclitaxel analogue Flutax-2 (17,27,28). These experiments are designed to address the question of whether Flutax-2 has a greater affinity for acetylated microtubules relative to deacetylated microtubules in vitro.…”

Section: Resultsmentioning

confidence: 99%

Farnesyltransferase Inhibitors Reverse Taxane Resistance

Marcus

O′Brate²,

Buey³

et al. 2006

Cancer Research

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The combination of farnesyltransferase inhibitors (FTIs) and taxanes has been shown to result in potent antiproliferative and antimitotic synergy. Recent phase I and II clinical trials have shown that this combination shows clinical activity in taxane-refractory or taxane-resistant cancer patients. To understand the mechanism behind these clinical observations, we used a cancer cell model of paclitaxel resistance and showed that the FTI/taxane combination retains potent antiproliferative, antimitotic, and proapoptotic activity against the paclitaxel-resistant cells, at doses where each drug alone has little or no activity. To probe the mechanistic basis of these observations, paclitaxel activity was monitored in living cells using the fluorescently conjugated paclitaxel, Flutax-2. We observed that all FTIs tested increase the amount of microtubule-bound Flutax-2 and the number of microtubules labeled with Flutax-2 in both paclitaxel-resistant and paclitaxel-sensitive cells. Importantly, we observed a consequential increase in microtubule stability and tubulin acetylation with the combination of the two drugs, even in paclitaxel-resistant cells, confirming that the enhanced taxane binding in the presence of FTI affects microtubule function. Furthermore, this mechanism is dependent on the function of the tubulin deacetylase, HDAC6, because in cells overexpressing a catalytically inactive HDAC6, FTIs are incapable of enhancing Flutax-2-microtubule binding. Similar results were obtained by using an FTI devoid of farnesyltransferase inhibitory activity, indicating that functional inhibition of farnesyltransferase is also required. Overall, these studies provide a new insight into the functional relationship between HDAC6, farnesyltransferase, and microtubules, and support clinical data showing that the FTI/taxane combination is effective in taxane-refractory patients. (Cancer Res 2006; 66(17): 8838-46)

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“…[14,15] From a structural perspective, the paclitaxel-binding site, located in the lumen of the microtubules, [17] is not easily accessible to the paclitaxel molecules in solution. However, it was also shown that paclitaxel binds very quickly to microtubules [18] and that a fluorescent tag attached to paclitaxel bound to microtubules can be recognized by an antibody, [19] which indicates an additional, and external, accessible binding site. This apparent paradox of a hidden but easily accessible luminal binding site was elucidated by the discovery of a new external binding site, [20] to which a covalently binding MSA, cyclostreptin, [12,21] binds before being internalized to the luminal site.…”

Section: Introductionmentioning

confidence: 99%

The Bound Conformation of Microtubule‐Stabilizing Agents: NMR Insights into the Bioactive 3D Structure of Discodermolide and Dictyostatin

Canales

Matesanz

Gardner

et al. 2008

Chemistry A European J

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Macromolecular Accessibility of Fluorescent Taxoids Bound at a Paclitaxel Binding Site in the Microtubule Surface

Abstract: The macromolecular accessibility of the paclitaxel binding site in microtubules has been investigated using a fluorescent taxoid and antibodies against fluorescein, which cannot diffuse into the microtubule lumen. The formation of a specific ternary complex of microtubules, Hexaflutax

Cited by 47 publications

References 53 publications

FRET measurements of kinesin neck orientation reveal a structural basis for processivity and asymmetry

FRET measurements of kinesin neck orientation reveal a structural basis for processivity and asymmetry

Farnesyltransferase Inhibitors Reverse Taxane Resistance

The Bound Conformation of Microtubule‐Stabilizing Agents: NMR Insights into the Bioactive 3D Structure of Discodermolide and Dictyostatin

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