Tubulin sequence region beta 155-174 is involved in binding exchangeable guanosine triphosphate.

A combination of ligand binding and sedimentation equilibrium studies was used to characterize the thermodynamic linkages between alpha beta tubulin association, nucleotide binding, and the interaction of colchicine analogues with dimeric and dissociated tubulins. The strength of binding of allocolchicine to the tubulin dimer was identical (8 x 10(5) M-1) whether the exchangeable nucleotide site (E site) was occupied by GTP or GDP. This drug bound to dimeric (alpha beta) tubulin and to one of the monomeric subunits, and the binding affinity for the dissociated state was linked to occupancy of the exchangeable nucleotide site. When the exchangeable site was occupied by GTP, the drug bound with very similar affinities to the dimeric and dissociated states of the protein. For tubulin-GDP, the binding of the drug to the dissociated state was significantly weaker (6.3 x 10(4) M-1) than to the dimeric state, suggesting the existence of an E-site-related conformational change in the dissociated state. Podophyllotoxin, which contains the A-ring portion of colchicine, bound with equal affinity to the dimeric and dissociated forms of both tubulin-GTP and tubulin-GDP, indicating that it is the C-ring portion of colchicine that is linked to the E-site-related conformational change.(ABSTRACT TRUNCATED AT 250 WORDS)

Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Effect of colchicine analogs on the dissociation of .alpha..beta. tubulin into subunits: The locus of colchicine binding

Shearwin¹,

Timasheff²

1994

“…We have undertaken to define the taxoid binding site by photoaffinity labeling, an excellent tool for the identification and characterization of ligand-specific mediators of many biological and pharmacological phenomena (Bayley & Staros, 1984;Schuster et al, 1989). This technique has been applied to identify the binding site of several drugs and ligands of tubulin such as vinblastine (Safa et al, 1987), colchicine (Williams etal., 1985;Hahn etal., 1992),rhizoxin (Sawada et al, 1993), and GTP (Hesse et al, 1987;Linse & Mandelkow, 1988). One study has been performed using direct photoaffinity labeling of tubulin by Taxol which indicated selective binding of Taxol to /3-tubulin (Rao et al, 1992).…”

mentioning

confidence: 99%

Predominant Labeling of .beta.- over .alpha.-Tubulin from Porcine Brain by a Photoactivatable Taxoid Derivative

Combeau¹,

Commerçon²,

Mioskowski³

et al. 1994

An [(azidophenyl)ureido]taxoid (TaxAPU) was synthesized in a radiolabeled form by coupling an aminotaxoid to tritiated N-methyl-N-(chloroformyl)-p-azidoaniline. TaxAPU was used to photolabel polymerized porcine brain tubulin. This newly synthesized probe possesses taxoid properties as demonstrated by its effect, in the absence of light, on the kinetics of tubulin assembly and microtubule disassembly and on the critical concentration of tubulin. TaxAPU apparently competes with Taxol for the same binding site with an equilibrium dissociation constant of 6 microM. The photoactivation of 266 nm of the radiolabeled probe in the presence of microtubules led to a covalent incorporation of radioactivity. Analysis of the radiolabeled polypeptides by electrophoresis under denaturing conditions revealed a specific incorporation of tritium in both the alpha-and beta-subunits of tubulin. A dependence on probe concentration was observed for the irreversible radioactivity incorporated into both subunits and maintained essentially a ratio of 2.5:1 between beta/alpha. Therefore, TaxAPU constitutes a true photoaffinity probe for the taxoid binding site on microtubules. Our results complement those reported by Rao et al. (1992) of photo-cross-linking experiments with unmodified Taxol.

“…Dissociation of the ß dimer was used to probe the nature of the nonexchangeable nucleotide site (N site). Incubation of tubulin, diluted to a level where -ß dissociation occurs, with a 5000-fold excess of GDP showed no evidence of nucleotide exchange at the N site, leading to the conclusion that GTP must occupy the N site with an affinity 106-107 times greater than that for the E site.Tubulin, the basic subunit component of microtubules, is itself composed of two similar but nonidentical subunits, a and ß, each of which can bind 1 equiv of guanine nucleotide.The binding site on one subunit, the exchangeable or E site,1 identified as the ¡3-subunit (Geahlen & Haley, 1977;Hesse et al, 1987), can freely exchange with exogeneous nucleotide, while the site located on the other subunit (nonexchangeable or N site) cannot. That an equilibrium exists between the a and ß subunits of tubulin has been well-established by a variety of procedures, including sedimentation equilibrium (Detrich…”

mentioning

confidence: 99%

“…The binding site on one subunit, the exchangeable or E site,1 identified as the ¡3-subunit (Geahlen & Haley, 1977;Hesse et al, 1987), can freely exchange with exogeneous nucleotide, while the site located on the other subunit (nonexchangeable or N site) cannot. That an equilibrium exists between the a and ß subunits of tubulin has been well-established by a variety of procedures, including sedimentation equilibrium (Detrich…”

mentioning

confidence: 99%

Linkages between the dissociation of .alpha..beta. tubulin into subunits and ligand binding: The ground state of tubulin is the GDP conformation

Shearwin

Pérez-Ramírez²,

Sn³

1994

The effects of ligands on the dissociation of the alpha beta tubulin dimer into the two subunits were investigated using calf brain tubulin. Sedimentation equilibrium studies showed a number of linkages. In the absence of magnesium in the medium, tubulin-GTP, tubulin-GDP, and tubulin with the exchangeable site unoccupied associate with essentially the same strength (K alpha beta = 1 x 10(7) M-1). This indicates that the ground state of tubulin (i.e., in the absence of magnesium) is not affected by occupancy of the exchangeable nucleotide binding site (E site). The alpha beta association is enhanced by magnesium ions. The association of tubulin with GDP in the E site is linked to the uptake of twice as many magnesium ions as that of tubulin with GTP in the E site. This suggests that magnesium binding is linked to an E-site-related conformational change. Consideration of the linkages between the binding of magnesium ions, E-site occupancy, and tubulin conformation in terms of the model [Howard, W. D., & Timasheff, S. N. (1986) Biochemistry 25, 8292-8300] in which the tubulin alpha beta dimer exists in an equilibrium between two conformations, a microtubule-forming ("straight") state favored by GTP and a double-ring-forming ("curved") state favored by GDP, leads to the conclusion that the ground state of tubulin is the ring-forming or "curved" conformation. Thus, in the absence of magnesium, the tubulin heterodimer exists in the ring-forming conformation, whether the E site is occupied by GTP or GDP.(ABSTRACT TRUNCATED AT 250 WORDS)