Identification of Cysteine 354 of β-Tubulin as Part of the Binding Site for the A Ring of Colchicine

Abstract

The colchicine analog 3-chloroacetyl-3-demethylthiocolchicine (3CTC) is a competitive inhibitor of colchicine binding to tubulin, binds to tubulin at 37°C, but not at 0°C, and covalently reacts with ␤-tubulin at 37°C, but not at 0°C, in a reaction inhibited by colchicine site drugs. The approximate intramolecular distance between the oxygen at position C-3 in 3CTC and the chlorine atom of the 3-chloroacetyl group is 3 Å. Using decylagarose chromatography, we purified ␤-tubulin that had reacted with 3-(chloromethyl-[ Tubulin interacts with a wide variety of small ligands, including divalent cations, guanine nucleotides, and antimitotic drugs. Perhaps because of its instability, its tendency to polymerize into pleomorphic structures, and/or its microheterogeneity, tubulin or tubulin-ligand complexes have not been crystallized. A polymeric site for paclitaxel (1), probably on the ␤-subunit (2, 3), has been defined in zinc-induced sheets of antiparallel protofilaments. Efforts to define ligand binding sites have generally involved induction of covalent interactions between tubulin and specific ligands. Usually photoreactive derivatives of the ligand were used (e.g. Refs. 2-5), but "direct photoaffinity" labeling, in which a covalent bond between ligand and tubulin forms under UV irradiation, has also been a valuable technique (6 -8). In most cases the target amino acids in the covalent interaction between protein and ligand were not identified, with localization of the reactive site limited to the tubulin subunit or a peptide fragment.No ligand has attracted more attention than colchicine (9), whose interaction with tubulin permitted the initial isolation of the protein (10). Photoaffinity (4, 5) and direct photoaffinity (7, 11) studies have provided evidence that both the ␣-and ␤-subunits participate in forming the binding site, as has genetic evidence (12-14). In only one of these studies (11) was label localized to specific peptide fragments of tubulin, and the region of colchicine that reacted with the protein was uncertain.We have taken a different approach from concern that use of bulky photoaffinity groups introduces a paradox into the technique. Adequate affinity of such compounds for tubulin implies that the reactive group has been introduced at a position in the ligand of secondary importance for the binding reaction. Thus, covalent reaction(s) may occur at some distance from the binding site itself. While this problem does not arise with the direct photoaffinity method, the precise ligand-peptide reaction has only been established with GTP (6). We decided to explore the use of small, chemically reactive substituents introduced at defined positions in colchicinoids and allocolchicinoids (15, 16). Schmitt and Atlas (17) had used an analog derivatized with the bromoacetyl group in the B ring side chain, but they found extensive nonspecific covalent reactions between their analog and tubulin. We used the isothiocyanate and chloroacetyl groups (16), and introduced them into the side chain, at position C-9 of t...

β-Tubulin C354 Mutations that Severely Decrease Microtubule Dynamics Do Not Prevent Nuclear Migration in Yeast

“…In our previous article we speculated that the mutation at C354 might produce a change in the structure of the tubulin dimer that could translate into stronger interprotofilament interactions in the microtubule (Gupta et al, 2001). This residue appears to be located at or near the colchicine binding site (Bai et al, 1996). It has been proposed that colchicine and other antimitotic drugs that modify microtubule dynamics mimic naturally occurring compounds that regulate microtubule function (Wilson and Jordan, 1995).…”

“…For example, chemical modification of cysteine residues results in inhibition of tubulin assembly in vitro (Ludueñ a and Roach, 1991) and GTP hydrolysis by tubulin (Mejillano et al, 1996). Cysteine residues can also be covalently cross-linked to the exchangeable site guanine nucleotide (Shivanna et al, 1993;Jayaram and Haley, 1994;Bai et al, 1999), to a colchicine analogue (Bai et al, 1996) and to other antimitotic agents (Bai et al, 1989;Shan et al, 1999). Previously, we used sitedirected mutagenesis to investigate the roles of the six cysteine residues in Saccharomyces cerevisiae in the structure and function of tubulin (Gupta et al, 2001).…”

Posttranslational modification of tubulin by palmitoylation: I. In vivo and cell-free studies.

“…Colcemid (an analogue of colchicine), podophyllotoxin, and nocodazole share a binding site on ,B-tubulin (Hoebeke et al, 1976;Cortese et al, 1977;Uppuluri et al, 1993;Bai et al, 1996). Recent evidence indicates that this binding site is part of the binding site for Taxol (Rao et al, 1994(Rao et al, , 1995.…”

“…As described above, GMPCPP-assembled microtubules were a substrate for palmitoylation, and I was not able to identify conditions that inhibited palmitoylation of GMPCPP-assembled microtubules without causing nonpolymerized palmitoylated tubulin to aggregate and pellet during separation of polymer and supernatant by centrifugation. Thus, it remains possible that (Hoebeke et al, 1976;Cortese et al, 1977;Uppuluri et al, 1993;Rao et al, 1994Rao et al, , 1995Bai et al, 1996). Therefore, the effect of Colcemid (a derivative of colchicine), podophyllotoxin, and nocodazole on cell-free palmitoylation of tubulin was examined.…”

Paclitaxel-resistant Human Ovarian Cancer Cells Have Mutant β-Tubulins That Exhibit Impaired Paclitaxel-driven Polymerization

“…Photolabeling with a colchicine analog demonstrates that the A-ring of colchicine interacts with Cys 354 (49). Rhizoxin photolabels a peptide containing residues 363-379 (50).…”