NONCOOPERATIVE BINDING OF THE MAP-2 MICROTUBULE-BINDING REGION TO MICROTUBULES

Microtubule-associated protein (MAP)-2 is a multi-domain cytoskeletal protein that copurifies with brain microtubules (MTs) through repeated cycles of warm polymerization and cold disassembly. Recent equilibrium binding studies of high molecular weight MAP-2ab to taxol-stabilized MTs suggest that the interactions are highly cooperative, as indicated by sigmoidal binding curves, non-linear Scatchard plots, and an apparent all-or-none response in MAP binding in titration experiments (Wallis, K. T., Azhar, S., Rho, M. B., Lewis, S. A., Cowan, N. J., and Murphy, D. B. (1993) J. Biol. Chem. 268, 15158-15167). To learn more about the mechanism of MAP-2 binding to MTs, we investigated the binding properties of bacterially expressed MT-binding region (MTBR) of bovine brain MAP-2. Scatchard plots of the binding data showed no evidence of cooperativity, as reflected by the linear plots of ($) over bar upsilon/[MTBR](free) versus ($) over bar upsilon. The stoichiometry was 1-1.1 mol of MTBR/mol of tubulin dimer, and the dissociation constant for the MTBR was 1.1 mu M. Bovine brain tau protein competitively inhibited MAP-2 binding, as evidenced by an increased K-d value for MTBR binding to MTs. Although the second repeat peptide m(2) (VTSKCGSLKNIRHRPGGG) is thought to play a dominant role in MAP-2 binding to MTs, a MTBR mutant (with m(2) replaced by the third octadecapeptide repeat m(3)) displays an K-d of 2.8 +/- 0.1 mu M and a stoichiometry of 0.9 +/- 0.05 mol of MTBR/mol of tubulin dimer. Another mutant with additional copies of the second repeat, designated by us as MTBR[m(12)m(2)m(32)], displayed noncooperative binding with a K-d of 0.53 +/- 0.05 mu M and a stoichiometry of 2.2 +/- 0.2 mol of mutant MTBR/tubulin dimer. Equilibrium sedimentation experiments demonstrated that the wild-type MTBR is monomeric, whereas MTBR[m(12)m(2)m(32)] self-associates to a stable dimer over the concentration range used in our MT binding studies. This finding indicates that only one of the two MT-binding sites on the dimer is probably linked to a microtubule at any given time.