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The mammalian microtubule-associated proteins (MAPs), MAP2, MAP4, and τ, are structurally similar and considered to be evolutionarily related. The primary structure of a nematode MAP, PTL-1, also reportedly resembles those of the MAPs, but only in a small portion of the molecule. In this study, we elucidated the overall domain organization of PTL-1, using a molecular dissection technique. Firstly, we isolated nematode microtubules and proved that the recombinant PTL-1 binds to nematode and porcine microtubules with similar affinities. Then, the recombinant PTL-1 was genetically dissected to generate four shorter polypeptides, and their microtubule-binding and assembly promoting activities were assessed, using porcine microtubules and tubulin. PTL-1 was found to consist of two parts, microtubule-binding and projection domains, with the former further divided into three functionally distinct subdomains. The molecular architecture of PTL-1 was proved to be quite analogous to its mammalian counterparts, MAP2, MAP4, and τ, strongly supporting their evolutionary relationships.

We previously used nuclear magnetic resonance (NMR) to analyze the structure of a synthetic tricosapeptide corresponding to an active site of microtubule-associated protein 4 (MAP4). To further the structural analysis, we have constructed a minimal active domain fragment of MAP4, encompassing the entire active site, and obtained its NMR spectra. The secondary structure prediction using partially assigned NMR data suggested that the fragment is largely unfolded. Two other independent techniques also demonstrated its unfolded nature, indicating that MAP4 belongs to the class of intrinsically disordered proteins (IDPs). The NMR spectra of the fragment-microtubule mixture revealed that the fragment binds to the microtubule using multiple binding sites, apparently contradicting our previous quantitative studies. Given that MAP4 is intrinsically disordered, we propose a mechanism in which any one of the binding sites is active at a time, which is one of the typical interaction mechanisms proposed for IDPs.

To analyze the interaction between microtubule-associated protein (MAP) 4 and microtubules physicochemically, a MAP4 active site fragment was designed for nuclear magnetic resonance (NMR) use. The fragment was bacterially expressed and purified to homogeneity. The buffer conditions for NMR were optimized to support microtubule assembly. The fragment was found to bind to microtubules under the optimized buffer conditions.