EXPLORING THE MICROTUBULE-BINDING REGION OF BOVINE MICROTUBULE-ASSOCIATED PROTEIN-2 (MAP-2) - CDNA SEQUENCING, BACTERIAL EXPRESSION, AND SITE-DIRECTED MUTAGENESIS
Rl. Coffey et al., EXPLORING THE MICROTUBULE-BINDING REGION OF BOVINE MICROTUBULE-ASSOCIATED PROTEIN-2 (MAP-2) - CDNA SEQUENCING, BACTERIAL EXPRESSION, AND SITE-DIRECTED MUTAGENESIS, Biochemistry, 33(45), 1994, pp. 13199-13207
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.