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Vinca alkaloids bind to the tubulin heterodimer on the beta subunit and induce a reversible self association into spiral polymers. The process has been extensively studied by sedimentation velocity studies in the laboratories of Serge Timasheff & more recently Jack Correia. The drugs induce an interacting system that sediments as a skewed boundary with a fast zone and a trailing centripetal zone. Our new studies use the absorbance optical system in the Beckman XLA and thus are conducted at 1-4 uM tubulin. This is well below the concentrations used by Timasheff & coworkers (50-200 uM in a schlieren optical system) and demonstrate these drugs have significant effects at physiologic concentrations. Titrations are performed from 0.1 to 70 uM drug and each sedimentation velocity data set is processed through DCDT to generate a g(s) which is converted to a weight average sedimentation coefficient. These data (drug concentration, protein concentration, Sw) are then fit to a ligand-mediated self-association scheme to extract the affinity of drug for tubulin and the liganded tubulin self association constant. Experiments conducted as a function of temperature reveal a positive enthalpy and entropy for spiral formation. In addition, experiments as a function of nucleotide content reveal an allosteric enhancement of self association induced by GDP relative to GTP. Spiral assembly is enhanced by divalent cation concentration but these cations also strongly promote lateral association and spiral condensation into sheets. The addition of NaCl suppresses this lateral association and suggests a significant electrostatic component to the lateral interaction. Surprisingly, unlike microtubule assembly, pH (6.3-7.5) has no effect on drug binding or self association. This suggests lateral protofilament interactions are the site of pH effects in microtubule assembly.
We have now compared eight vinca alkaloids by sedimentation velocity studies and have demonstrated a definitive order of spiral tendency that correlates with the cytotoxic action and the clinical dose of each drug. [Vincrisitine > Vinepidine > Vindesine > Vinblastine > Vinorelbine > Desformyl Vincrisitine > Desacetyl Desformyl Vincristine > Vinflunine} Each drug exhibits the same GDP allosteric effect and the order of overall affinity (K1K2) is identical in different nucleotide and ionic conditions. These data strongly suggest that the mechanism of action of these drugs is mediated by the extent of spiral assembly, and since the drugs acts on microtubules directly, it suggests spiral assembly occurs from the ends of microtubules. A direct measure of spiral interaction with the ends of microtubules is our current focus. Vinca alkaloid induced spirals are equilibrium polymers that exhibit slow relaxation kinetics upon dilution. This would account for the initial stabilization of microtubules that suppresses dynamic instability.
Correia, J.J. and Lobert. S. Sedimentation Velocity Studies on the Interaction of Dolastatin 10 with PC-tubulin. Submitted.
Investigations into the mode of action of microtubule based molecular motors has extensively utilized constructs of these molecules that retain microtubule and ATP binding ability but lack the cargo domain. An important question is the state of assembly of these constructs since the mechanism of motility reportedly requires two motor domains in a dimer acting in a "hand-over-hand" manner. We have thus established collaborations with three laboratories to measure the molecular weight of kinesin and ncd motor domain constructs and where possible to measure the dimerization constant for there reversible self association. An additional complexity is the tendency of these constructs to irreversibly aggregate, especially if they are His tagged.
We are currently developing methods to study the interact of molecular motors with tubulin heterodimers. These experiments are important because they independently verify the affinity of motor domains, particularly single head or monomeric constructs, for tubulin, and allow for the dissection of cooperative interactions and the dependence upon both GXP and AXP nucleotides in the absence of processive ATP hydrolysis. Sedimentation velocity offers a rapid method for observing the formation of complexes formed between kinesin or ncd motor domains and tubulin under nonmicrotubule forming conditions. The shapes of the g(s) patterns can be simulated, by methods developed by Walter Stafford at BBRI, to establish the strength of the interactions. Walter has also developed a fitter, ABCDfitter, that fits concentration difference curves (to eliminate systematic baseline noise) to a model that includes 1:1 and 2:1 complex formation. We are currently working with a human kinesin construct, K420, provided by Ron Vale, to probe these interactions. A manuscript describing this work is in preparation.
This approach for studying hetero interactions by sedimentation velocity has been generalized by the program SedAnal developed by Walter Stafford at BBRI. In this case the formation of a 1 op18:2 tubulin heterodimer complex has been studied as a function of pH (6.8 vs 7.5) and nucleotide content (GDP vs GTP). The reaction is favored by low pH and the presence of GDP.
In collaboration with Kai Lin at Univ of Mass. Medical School, we are investigating the ability of smad3 and smad4 to form trimers and hetero-trimers. The role of receptor activated phosphorylation is a major focus of this work. Current studies are dissecting the energetic contribution of phosphorylation at each of three sites (SSVS) on homo- and hetero-trimerization. Additional studies have now been performed on similar smad1 constructs. Phosphorylation mimics (SDVD) greatly enhance trimerization and crystallization confirms the interaction of the C-terminal phosphorylation domain with adjacent subunits in an asymmetric manner, consistent with the formation of 2:1 hetero-trimers.
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