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A new class of microtubule-stabilizing agents, the epothilones, has been identified. Epothilones induce tubulin (TB) polymerization in the absence of GTP and cause microtubule stabilization and bundling. The Glutamine (Gln) to Glutamic Acid (Glu) mutation at residue 292 (Gln292Glu) situated near the M-loop of b-tubulin affects Epothilone A (EpoA) with a resistance of 72-fold. Through the minimization/MD simulation process, it is our intent to show a chain effect that causes the Gln292Glu mutation to effect the binding of EpoA. The terminal amide of this Gln residue hydrogen bonds to the Leu273 which is adjacent to Thr274, the amino acid residue in direct contact with the ligand. The electrostatic repulsion occasioned by the negative carboxylate of the Glu292 can be expected to reorganize this pocket and translate its effects outward to Thr274. Moreover, the negatively charged side chain of Glu292 also increases the hydrophilicity of this region, as observed by more water molecules being placed and a stronger hydrogen-bonding network formed between Glu292 and the Leu273 backbone. A reasonable hypothesis of the observed 72-fold EpoA resistance is that the network of hydrogen bonds associated with polar groups from C-3 to C-7 of the epothilone is perturbed sufficiently, so that ligand binding is considerably weakened. After a series of MD Simulations at 20K and 100K the wild-type and mutant-type were compared in the gas and water phases to examine the hydrogen bonding patterns. The interaction patterns of the residues connecting the Gln292Glu mutation and EpoA were disturbed through both phase simulations. EpoA begins to move slightly out of its pocket at 20K and 100K as a result of the hypothesized chain effect. In addition, the Gln292Glu mutation appears to cause significant movement of the secondary structure at the M-loop between helices 6 and 7 in comparison to the wild-type backbone. This foreshadows further experimentation at 200K and 300K to explain the 72-fold resistance of EpoA to the Gln292Glu mutation.
*Microtubules are major dynamic structural components in cells that are essential for the development and maintenance of cell shape, cell signaling, movement, and division. *Microtubules are cytoskeletal polymers built by self association of αβ-tubulin dimers that exist in a state of constant dynamic equilibrium between the polymer and its soluble dimer form.
*Constant shortening and elongation is necessary for microtubules to function within a cell.
*Drugs that bind to either tubulin or microtubules correspond to two of the most effective classes of anticancer agents.
*Although different classes of anti-tubulin drugs bind to distinct sites on the tubulin dimer or on microtubules, they all affect microtubule dynamics, block mitosis at the metaphase/anaphase transition and consequently induce cell death.
*A new class of microtubule-stabilizing agents, the epothilones, has recently been identified.
*Epothilones induce tubulin polymerization in the absence of GTP and causes microtubule stabilization and bundling.
*Gln292Glu on b-tubulin affects epo-A with a resistance of 72-fold.
The structure of EpoA, bound to αβ-TB in zince-stabilized sheets was determined by a combination of electron crystallography at 2.89Å resolution and nuclear magnetic resonance-based conformational analysis.
Step I. Mutate Residue: Convert the glutamine292 residue to a charged glutamic acid residue.
Step II. Minimization: What is the most preferable conformation for the Glu292?
Step III. MD Simulation
Step IV. MD Simulation with Water Molecules: Place buried waters into the hydrophobic cavity and solvate the complex DOWSER PROGRAM: by Jan Hermans3 (http://hekto.med.unc.edu:8080/HERMANS/software/DOWSER/Dowser.html) SOLVATE PROGRAM: by Helmut Grubmüller (http://www.mpibpc.gwdg.de/abteilungen/071/solvate/docu.html) Follow the movement of EpoA and its surrounding pocket using SYBYL7.0.
*Tubulin side chains linking residue 292 and Epothilone A appear to experience greater loss of hydrogen bonding for the Gln292Glu mutant than for the wild-type when examined by MD simulation in both the gas and water phases.
*Gas Phase - Since there is minimal stability within this phase, the results depend on the amount of time the WT and MT spend in the b-tubulin binding pocket. At 20K a difference can be monitored because the temperature is not high enough to force the EpoA out of its binding site in either case. The WT maintains most of its interactions as does the MT. However, most of the MT atoms forming hydrogen bonds experience significantly larger separations demonstrating that these interactions are being lost.
*Water Phase - Exemplifies the loss of hydrogen bonding just as for the gas phase. In addition, however, a notable change in the secondary backbone structure of the αβ-tubulin is evident because the water molecules attempt to form a stronger hydrogen bonding network by incorporating more H2O. This changes the conformation of both the M and H6-H7 loops.
*EpoA begins to move slightly out of its pocket by means of the chain effect hypothesized at 20K. Further experimentation at 100, 200K and 300K is needed to confirm that the dynamic changes observed for the mutant tubulin are faithfully reproduced at higher T.
This material is based upon work supported be the Howard Hughes Medical Institute under Grant No.52003727 and the Cherry Emerson Fellowship program.
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