Authors
S Sajko2; I Grishkovskaya2; M Puchinger2; J Kostan2; B Morriswood1; K Djinovic-Carugo2; 1 Department of Cell & Developmental Biology, Biocentre, University of Würzburg, Würzburg, Germany, Germany; 2 Department of Structural and Computational Biology, Max F. Perutz Laboratories, University of Vienna, Austria Discussion
Membrane occupation and recognition nexus repeat (MORN)-containing proteins are found throughout the tree of life. The MORN1 protein in Trypanosoma brucei is composed of 15 MORN repeats and is essential for the viability of the parasite`s bloodstream form. It localizes to a hook-shaped complex that wraps around the neck of the flagellar pocket membrane. Our biophysical characterization of TbMORN1 including circular dichroism, electron microscopy, and solution small angle X-ray scattering (SAXS) suggest that it is an all-beta protein, existing in a rod-shaped dimer. These data are consistent with a recently solved crystal structure for TbMORN1 homologue from Plasmodium falciparum, the construct missing the first 6 MORN repeats. Mutational, chemical cross-linking, and SAXS data indicate that TbMORN1 dimerizes via its C-terminal repeats. Lipid blots, native electrophoresis, and fluorescence anisotropy assays showed that TbMORN1 binds to the lipid phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2) with low micromolar affinity. Based on a computational model, putative PI(4,5)P2 binding sites were predicted and PI(4,5)P2 binding mutants have been generated and assayed. With the ongoing structural and in vivo studies we are trying to understand whether PI(4,5)P2-binding is essential for correct protein assembly and/or localization in the parasite. 
