Sanchez-Pulido L et al. (2016), The C-terminal domain of TPX2 is made of alpha-...

XB-ART-54008

BMC Struct Biol 2016 Oct 26;161:17. doi: 10.1186/s12900-016-0070-8.

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The C-terminal domain of TPX2 is made of alpha-helical tandem repeats.

Sanchez-Pulido L , Perez L , Kuhn S , Vernos I , Andrade-Navarro MA .

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BACKGROUND: TPX2 (Targeting Protein for Xklp2) is essential for spindle assembly, activation of the mitotic kinase Aurora A and for triggering microtubule nucleation. Homologs of TPX2 in Chordata and plants were previously identified. Currently, proteins of the TPX2 family have little structural information and only small parts are covered by defined protein domains. METHODS: We have used computational sequence analyses and structural predictions of proteins of the TPX2 family, supported with Circular Dichroism (CD) measurements. RESULTS: Here, we report our finding that the C-terminal domain of TPX2, which is responsible of its microtubule nucleation capacity and is conserved in all members of the family, is actually formed by tandem repeats, covering well above 2/3 of the protein. We propose that this region forms a flexible solenoid involved in protein-protein interactions. Structural prediction and molecular modeling, combined with Circular Dichroism (CD) measurements reveal a predominant alpha-helical content. Furthermore, we identify full length homologs in fungi and shorter homologs with a different domain organization in diptera (including a paralogous expansion in Drosophila). CONCLUSIONS: Our results, represent the first computational and biophysical analysis of the TPX2 proteins family and help understand the structure and evolution of this conserved protein family to direct future structural studies.

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Species referenced: Xenopus
Genes referenced: kif15 tpx2

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	Fig. 1. Phylogenetic trees of TPX2 homologs. a Phylogenetic tree of full length orthologs of TPX2 in representative species. b Phylogenetic tree of short orthologs of TPX2 in representative dipteran species. Drosophila has three paralogs. The labels indicate species and length of the protein. Numbers in the tree represent bootstrapping values. The sequences and NCBI identifiers are available as Additional file 1 and Additional file 3 for (a) and (b), respectively. The multiple sequence alignments used to do the phylogenetic trees are available as Additional file 2 and Additional file 4 for (a) and (b), respectively
	Fig. 2. Repeats in TPX2 proteins. a Multiple sequence alignment of tandem repeats in Xenopus laevis, human and A. thaliana TPX2. The red box indicates a summary of predictions for an alpha-helix (see Methods for details). b Position of repeats in human TPX2. UniProt database identifiers are Q6NUF4 for xlTPX2, Q9ULW0 for TPX2_HUMAN, and F4I2H7 for atTPX2. The multiple sequence alignment is available as Additional file 5
	Fig. 3. Biochemical and structural analysis of TPX2. a SDS-PAGE analysis of Xenopus and Arabidopsis TPX2 proteins. b Spectra in the region of 260â190Â nm were obtained at 25Â Â°C for full length xlTPX2 and atTPX2. Both spectra present a typical alpha helical profile with two minima (Î»208 and Î»222 nm). c Molecular model of xlTPX2 (Q191-K715) represent a compact structure of repeated Î±-helices linked by aÂ flexible loop. d Ramachandran plot ofÂ the xlTPX2 model. About 96Â % of all residues were in favored regions, and about 4 % of the residues were in an allowed region. Two outliers were found, Leucines at positions 173 and 302, although, visual inspection did not reveal any steric clash

References [+] :

Adamczak, Combining prediction of secondary structure and solvent accessibility in proteins. 2005, Pubmed