EXPLORING THE MICROTUBULE-BINDING REGION OF BOVINE MICROTUBULE-ASSOCIATED PROTEIN-2 (MAP-2) - CDNA SEQUENCING, BACTERIAL EXPRESSION, AND SITE-DIRECTED MUTAGENESIS

A 1.1 kilobase fragment of bovine microtubule-associated protein-2 (MA P-2) cDNA coding for bovine MAP-2 microtubule-binding region (MTBR) wa s sequenced. Relative to mouse, rat, and human MAP-2, we observed stri king preservation of primary structure, even beyond the sequence and s pacing of the three nonidentical peptide repeats responsible for micro tubule-binding interactions. For further analysis of microtubule-MAP i nteractions using site-directed mutagenesis, we developed a bacterial expression system coding for the MT-binding fragment of MAP-2 starting at the thrombin cleavage site (position 1629) and continuing to the C -terminus. This MT-binding fragment was purified to homogeneity by tak ing advantage of the unusual heat-stability and isoelectric properties of this cytomatrix component. We found that the MT-binding domain rea dily promoted tubulin polymerization, and the critical tubulin concent ration was reduced in the presence of this recombinant protein. Becaus e a second repeated sequence analogue can promote tubulin polymerizati on as well as displace the MT-binding region of MAP-2, this study was designed to learn more about the importance of each repeated sequence in MT binding. Accordingly, we mutated the first and third sequences t o resemble the second repeated sequence, thereby generating the mutant s designated m(12)-m(2)-m(3), m(1)-m(32), and m(12)-m(2)-m(32). These recombinant proteins bound with an affinity comparable to or slightly better than equal concentrations of wild-type MT-binding fragment. Lik ewise, when the first or third sequence was replaced by an exact copy of the second octadecapeptide repeat, there was little, if any, increa se in binding affinity, as reflected in the ability of mutant MT-bindi ng fragments to promote tubulin polymerization. We can thus conclude t hat the binding energies associated with each of the three second-sequ ence repeats were not additive, and these findings fortify the conclus ion that the second sequence repeat plays a dominant role in microtubu le binding.