Ricardo B. Maccioni

A discrete repeated sequence defines a tubulin binding domain on microtubule-associated protein tau

The protein domain responsible for the interaction of tau with tubulin has been identified. Biophysical studies indicated that the synthetic peptide Val187-Gly204 (VRSKIG-STENLKHQPGGG) from the repetitive sequence on tau binds to two sites on the tubulin heterodimer and to one site on each of the microtubule-associated protein-interacting C-terminal tubulin peptides alpha(430-441) and beta(422-434). The binding data showed a relatively stronger interaction of Val187-Gly204 with beta(422-434) as compared to that with alpha(430-441). The interaction of this tau peptide with either alpha or beta tubulin peptides appears to be associated with conformational changes in both the tau and the tubulin peptides. The beta tubulin peptide also appears to induce a structural change of tau fragment Val218-Gly235. Interestingly, tau peptides Val187-Gly204 and Val218-Gly235 induced tubulin self-assembly in a cold-reversible fashion, and incorporated into the assembled polymers. The specificity of the interaction of the tau peptide was supported by the competition of tau protein for the interaction with the tubulin polymer. In addition, the tau peptide appears to contain the principal antigenic determinant(s) recognized by anti-idiotypic antibodies that react with the tubulin binding domains on microtubule-associated proteins. The present findings together with the demonstration of the presence of multiple sites for the binding of the alpha(430-441) and beta(422-434) tubulin fragments to tau, and the existence of repetitive sequences on tau, strongly support the hypothesis that the region of tau defined by the repetitive sequences is involved in its interaction with tubulin.

Heat-stable microtubule protein MAP-1 binds to microtubules and induces microtubule assembly

The microtubule‐associated proteins, MAP‐1, MAP‐2 and tau, have been purified from brain tissue via a new approach using a heating step directly on the homogenate, followed by selective adsorption on calmodulin‐Sepharose affinity columns and gel‐filtration chromatography. Our results indicate that these MAPs share common biochemical properties, including heat stability, calmodulin binding and promotion of tubulin assembly into microtubules.

Small zone gel chromatography of interacting systems: Theoretical and experimental evaluation of elution profiles for kinetically controlled macromolecule-ligand reactions

A phenomenological theory of small zone gel chromatography is elaborated for kinetically controlled macromolecule-ligand interactions. Chromatography, direct binding experiments, and rate measurements are used for successful comparison of experimental behavior with theoretical elution profiles for the interaction of MAP-2 with two synthetic peptide fragments from the C-terminal moieties of alpha- and beta-tubulin subunits. The results of this study provide guidelines for interpretation of experimental small zone elution profiles of total ligand.

Common antigenic determinants of the tubulin binding domains of the microtubule-associated proteins MAP-2 and tau

The structural-functional aspects of the tubulin binding domain on the microtubule-associated protein MAP-2, and its relationship with the tubulin binding domain on tau, were studied using anti-idiotypic antibodies that react specifically with the epitope(s) on MAPs involved in their interaction with tubulin in addition to other tau and MAP-2 specific antibodies. Previous studies showed that MAP-2 and tau share common binding sites on tubulin defined by the peptide sequences alpha (430-441) and beta (422-434) of tubulin subunits. Furthermore, binding experiments revealed the existence of multiple sites for the interaction of the alpha- and beta-tubulin peptides with MAP-2 and tau. Most recent studies showed that the synthetic tau peptide Val187-Gly204 (VRSKIGSTENLKHQPGGG) from the repetitive sequence on tau defines a tubulin binding site on tau. Our present immunological studies using anti-idiotypic antibodies which interact with the synthetic tau peptide and antibodies against the Val187-Gly204 tau peptide indicate that MAP-2 and tau share common antigenic determinants at the level of their respective tubulin binding domains. These antigenic determinants appear to be present in the 35 kDa tubulin binding fragment of MAP-2 and in 18-20 kDa chymotryptic fragments containing the tubulin binding site(s) on MAP-2. These findings, along with structural information on these proteins, provide strong evidence in favor of the hypothesis that tubulin binding domains on MAP-2 and tau share similar structural features.

A Proton Magnetic Resonance and a Circular Dichroism Study of the Solvent Dependent Conformation of the Synthetic Tubulin Fragment Ac Tubulin, Alpha (430–441) Amide and its Interaction with Substance-P

Proton magnetic resonance techniques were used to study the conformation of the synthetic tubulin fragment Ac-tubulin (430-441) amide in H2O and 80% CD3OH/20% D2O solutions, using water suppression techniques. Proton assignments are based on two-dimensional COSY experiments combined with one-dimensional spin decoupling. A comparison of the NH proton shifts between the two solvents, namely delta(CD3OH/H2O-H2O) shows a small solvent effect for the Lys1 to Val6 region of the molecule, whereas for Gly7 to Glu12 the solvent effect is much larger. The smaller effects in the region of Lys1 to Val6 may be due to some hydrogen bonding as these protons are shielded from the solvent. These conclusions are in agreement with the circular dichroism results in 80% methanol/20% water where the alpha helix is present to the extent of 30%, whereas the peptide is completely unstructured in water with some aggregation. The temperature dependence of the NH proton shifts was also carried out. In water these shifts are of the order of 7-9 X 10(-3) ppm/K indicating that most of the protons are not involved in hydrogen bonding. In CD3OH/H2O, these values range from about 4-6 X 10(-3) ppm/K, which are compatible with the presence of hydrogen bonds. Finally, binding studies were carried out between the tubulin peptide and the undecapeptide neutrotransmitter substance P. The largest shifts are for the Tyr3 NH proton of the tubulin fragment, whereas for substance P it is for the Lys3, Gln5 and Leu10 NH protons, indicating a change in conformation of both peptides on interaction.

Microtubule assembly affected by the presence of denatured tubulin.

Abstract The effects of denatured tubulin on microtubule assembly from active phosphocellulose-tubulin have been studied. The presence of denatured tubulin resulted in an inhibition of the assembly and in the increase of the critical concentration to trigger the assembly. Inhibition of both the rate and extent of microtubule assembly was dependent on denatured tubulin concentration. This perturbation of microtubule assembly by denatured tubulin is likely to be specific as non-microtubule proteins did not significantly affect the assembly.

Regulatory aspects of the colchicine interactions with tubulin

Limited proteolysis of tubulin with subtilisin results in the cleavage of both tubulin subunits yielding S-tubulin heterodimer and 4 kDa peptide fragments containing the carboxyl-terminal domains of α- and β-polypeptide chains. S-tubulin binds colchicine and the characterization of the binding of colchicine to S-tubulin molecules showed a decreased rate of decay of colchicine binding activity as compared to that of undigested tubulin. However, S-tubulin exhibited a lower colchicine binding constant than tubulin. Peptide fragments resulting from the controlled tryptic proteolysis of both pure tubulin and S-tubulin were purified by filtration chromatography and presented a strong colchicine binding activity with association constants of 4.5×106 and 2.7×106 M−1, respectively. Furthermore, these studies support our initial findings on the localization of the tubulin site for colchicine (Serrano L, Avila J, Maccioni RB: J Biol Chem 259:6607–6611, 1984) and define the colchicine binding domain in a domain of α-subunit from the point of limited tryptic cleavage to the site of subtilisin controlled proteolysis of that tubulin subunit. On the basis of these alterations in the interaction of colchicine upon removal of the C-terminal moiety of tubulin and since no change in the number of binding sites was found after subtilisin digestion, we suggest that the carboxyl-terminal region of tubulin subunits modulates the binding of colchicine.