Distances between the Paclitaxel, Colchicine, and Exchangeable GTP Binding Sites on Tubulin ² Yi Han, ‡ Henryk Malak, § A. G. Chaudhary, | M. D. Chordia, | David G. I. Kingston, | and Susan Bane* ,‡ Department of Chemistry, State UniVersity of New York at Binghamton, Binghamton, New York 13902-6016, Center for Fluorescence Spectroscopy, UniVersity of Maryland School of Medicine, Baltimore, Maryland 21201, and Department of Chemistry, Virginia Polytechnic Institute and State UniVersity, Blacksburg, Virginia 24061-0212 ReceiVed August 11, 1997; ReVised Manuscript ReceiVed NoVember 25, 1997 ABSTRACT: Distances between the paclitaxel, colchicine, and exchangeable GTP binding sites on tubulin polymers have been probed using fluorescence spectroscopy. Techniques for measuring fluorescence resonance energy transfer (FRET) between fluorescent or chromophoric ligands for each binding site were employed. 2-Debenzoyl-2-(m-aminobenzoyl)paclitaxel (2-AB-PT) was the fluorophore ligand for the paclitaxel binding site; thiocolchicine, allocolchicine, and MDL 27048 were probes for the colchicine site, and 2′(or 3′)-O-(trinitrophenyl)guanosine 5′-triphosphate (TNP-GTP) was the fluorophore ligand for the exchangeable GTP site. The distance between the colchicine and paclitaxel binding sites was determined with two different acceptor ligands in the colchicine site. An average distance distribution of 17 Å was found in both cases. Energy transfer between 2-AB-PT bound to the paclitaxel site and TNP-GTP (acceptor) bound to the exchangeable GTP site was observed in the polymer. The average distance distribution between the fluorophores was 16.0 Å, but the half-width of the distribution was large (17.9 Å), which indicates that energy transfer between more than one donor-acceptor pair occurred in the system. One interpretation of this result is that 2-AB-PT serves as an energy transfer donor for two GTP sites, one contained on the same subunit and one on an adjacent protofilament. No FRET was observed between ligands bound to the colchicine and exchangeable GTP sites, indicating that the result of colchicine binding on the GTP region of -tubulin is a long range, allosteric effect. The results from these experiments are interpreted in terms of known structural features of microtubules. Microtubules are an integral part of the cytoskeleton of all eukaryotic cells. These structures participate in a vast number of cellular functions, including mitosis, morphogen- esis, intracellular transport, and secretion (1). The cylinder lattice of the microtubule is composed entirely of the protein tubulin, which is a highly conserved heterodimeric protein. Microtubules are formed by the reversible assembly of soluble tubulin into polymers and possess in vivo additional proteins such as microtubule-associated proteins and motor proteins on the surface of the lattice. Other proteins are not required for microtubule assembly in vitro; normal micro- tubules can be produced from purified tubulin under certain conditions (2). The pathway from soluble tubulin to the functional microtubule polymer is remarkably sensitive to endogenous and exogenous effectors (3). As a result, tubulin has been an important target for chemotherapy, with particular success as a receptor for antineoplastic agents. Drugs such as paclitaxel (Taxol), vincristine, and estramustine are widely used in cancer chemotherapy, and new tubulin-active drugs, such as the cryptophycins, have progressed to clinical testing (4-6). It appears that all these drugs interact with tubulin at or in the vicinity of only three binding sites, known as the colchicine, Vinca, and paclitaxel binding sites (7). Drug binding to the each site produces effects on gross polymer morphology that tend to be characteristic of the binding site involved. For example, most ligands that bind to the colchicine site on tubulin associate with the protein prior to its polymerization and subsequently inhibit assembly (8), while ligands that bind to the paclitaxel site on tubulin stimulate assembly and bind preferentially to tubulin poly- mers (9, 10). Drug binding to the Vinca site inhibits tubulin assembly at low concentrations, but at higher concentrations induces tubulin self-association into nonmicrotubule struc- tures and into paracrystals (11). The drug binding sites also appear to be linked to some degree. Paclitaxel will promote tubulin assembly into microtubules in the presence of calcium and in absence of GTP, 1 and the resulting polymers are cold-stable. If colchi- cine is bound to tubulin prior to treatment with paclitaxel, ² This work was supported in part by National Institutes of Health Grants CA55131 and CA48974 (to D.G.I.K.) and National Science Foundation Grant MCB9406424 (to S.B.). * To whom correspondence should be addressed. ‡ State University of New York at Binghamton. § University of Maryland School of Medicine. | Virginia Polytechnic Institute and State University. 1 Abbreviations: 2-AB-PT, 2-debenzoyl-2-(m-aminobenzoyl)pacli- taxel; DMSO, dimethyl sulfoxide; MDL 27048, trans-1-(2,5-dimethox- yphenyl)-3-[4-(dimethylamino)phenyl]-2-methyl-2-propen-1-one; EGTA, ethylene glycol bis(-aminoethyl ether)-N,N,N′,N′-tetraacetic acid; GTP, guanosine 5′-triphosphate; PIPES, piperazine-N,N′-bis(2-ethanesulfonic acid); PME1 buffer, 50 mM PIPES, 0.5 mM MgSO 4, and 1 mM EGTA at pH 6.9; PME2 buffer, 100 mM PIPES, 1 mM MgSO4, and 2 mM EGTA at pH 6.9; PMEG buffer, 100 mM PIPES, 1 mM MgSO4,2 mM EGTA, and 0.1 mM GTP at pH 6.9. 6636 Biochemistry 1998, 37, 6636-6644 S0006-2960(97)01976-4 CCC: $15.00 © 1998 American Chemical Society Published on Web 04/28/1998