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Tubulin polymerization promoting protein/p25 (TPPP/p25) modulates the dynamics and stability of the microtubule system and plays crucial role in the myelination of oligodendrocytes. Here we showed by CD, fluorescence, and NMR spectroscopies that Zn2+ is the first ligand that induces considerable rearrangement of the disordered TPPP/p25. Zinc finger motif (His2Cys2) (His61-Cys83) was identified within the flexible region of TPPP/p25 straddled by extended unstructured N- and C-terminal regions. The specific binding of the Zn2+ to TPPP/p25 induced the formation of molten globule but not that of a well-defined tertiary structure. The Zn2+-induced partially folded structure accommodating the zinc binding motif is localized at the single Trp76-containing region as demonstrated by fluorescence resonance energy transfer and quenching experiments. We showed that the Zn2+-induced change in the TPPP/p25 structure modified its interaction with tubulin and GTP coupled with functional consequences: the TPPP/p25-promote...

The disordered Tubulin Polymerization Promoting Protein/p25 (TPPP/p25) modulates the dynamics and stability of the microtubule system and plays a crucial role in differentiation of oligodendrocytes. Here we first demonstrated by multinuclear NMR that the extended disordered segments are localized at the N- and C-terminals straddling a flexible region. We showed by affinity chromatography, fluorescence spectroscopy and circular dichroism that GTP binds to TPPP/p25 likely within the flexible region; neither positions nor intensities of the peaks in the assigned terminals were affected by GTP. In addition, we demonstrated that TPPP/p25 specifically hydrolyses GTP in an Mg2+-dependent manner. The GTPase activity is comparable with the intrinsic activities of small G proteins suggesting its potential role in multiple physiological processes.

TPPP/p25 is a brain-specific protein, which induces tubulin polymerization and microtubule (MT) bundling and is enriched in Lewy bodies characteristic of Parkinson's disease [Tirián et al. (2003) Proc. Natl. Acad. Sci. U.S.A. 100, 13976-13981]. We identified two human gene sequences, CG1-38 and p25beta, which encoded homologous proteins, that we termed p20 and p18, respectively. These homologous proteins display 60% identity with tubulin polymerization promoting protein/p25 (TPPP/p25); however, the N-terminal segment of TPPP/p25 is missing. They could be clustered into three subfamilies present in mammals and other vertebrates. We cloned, isolated, and characterized the structural and functional properties of the recombinant human proteins at molecular, ultrastructural, and cellular levels using a number of tools. These data revealed that, while p20 behaved as a disorganized protein similarly to TPPP/p25, which was described as a flexible and inherently dynamic protein with a long unstructured N-terminal tail, p18 was featured in more ordered fashion. TPPP/p25 and p20 specifically attached to MTs causing MT bundling both in vitro and in vivo; p18 protein did not cross-link MTs, and it distributed homogeneously within the cytosol of the transfected HeLa cells. These data indicate that the two shorter homologues display distinct structural features that determine their associations to MTs. The properties of p20 resemble TPPP/p25. The bundling activity of these two proteins results in the stabilization of the microtubular network, which is likely related to their physiological functions.

article Background: The disordered Tubulin Polymerization Promoting Protein/p25 (TPPP/p25) modulates the dy- namics and stability of the microtubule system. In this paper the role of dimerization in its microtubule- related functions is established, and an approach is proposed to evaluate thermodynamic constants for mul- tiple equilibrium systems from ITC measurements. Methods: For structural studies size exclusion chromatography, SDS-PAGE, chemical cross-linking, circular di- chroism, fluorescence spectroscopy and isothermal titration calorimetry were used; the functional effect was analyzed by tubulin polymerization assay. Numerical simulation of the multiple equilibrium was performed with Mathematica software. Results: The dimerization of TPPP/p25 is promoted by elevation of the protein concentration and by GTP ad- dition. The dimeric form displaying enhanced tubulin polymerization promoting activity is stabilized by di- sulfide bond or chemical cross-linking. The GTP binding to the dimeric form (Kd-GTP = 200 μM) is tighter with one order of magnitude than to the monomeric one leading to the enrichment of the dimers. A mathe- matical model elaborated for the multiple equilibrium of the TPPP/p25-GTP system was validated by fitting the GTP-dependent changes of ellipticity and fluorescence signal in the course of TPPP/p25 titrations. The evaluation of the equilibrium constants rendered it possible to determine the thermodynamic parameters of the association of different TPPP/p25 forms with GTP from ITC measurements. Conclusions/General Significance: The dimerization of TPPP/p25 with favorable physiological functional poten- cy is proposed to play significant role in the fine tuning of TPPP/p25-mediated microtubule assembly; the un- folded monomers might be involved in the formation of pathological inclusions characteristic for Parkinson's disease and other synucleinopathies.

Tubulin polymerization promoting protein/p25 (TPPP/p25) modulates the dynamics and stability of the microtubule system and plays crucial role in the myelination of oligodendrocytes. Here we showed by CD, fluorescence, and NMR spectroscopies that Zn(2+) is the first ligand that induces considerable rearrangement of the disordered TPPP/p25. Zinc finger motif (His(2)Cys(2)) (His(61)-Cys(83)) was identified within the flexible region of TPPP/p25 straddled by extended unstructured N- and C-terminal regions. The specific binding of the Zn(2+) to TPPP/p25 induced the formation of molten globule but not that of a well-defined tertiary structure. The Zn(2+)-induced partially folded structure accommodating the zinc binding motif is localized at the single Trp(76)-containing region as demonstrated by fluorescence resonance energy transfer and quenching experiments. We showed that the Zn(2+)-induced change in the TPPP/p25 structure modified its interaction with tubulin and GTP coupled with functional consequences: the TPPP/p25-promoted tubulin polymerization was increased while the TPPP/p25-catalyzed GTPase activity was decreased as detected by turbidimetry and by malachite green phosphate release/(31)P NMR assays, respectively. The finding that the Zn(2+) of the bivalent cations can uniquely influence physiological relavant interactions significantly contributes to our understanding of the role of Zn(2+)-related TPPP/p25 processes in the differentiation/myelination of oligodendrocytes possessing a high-affinity Zn(2+) uptake mechanism.