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Structural base for the transfer of GPI-anchored proteins into fungal cell walls
Fungi, such as the unicellular model organism Saccharomyces cerevisiae, possess a thick cell wall composed of polysaccharides and proteins, which is essential for viable and healthy cells. While the mere synthesis of its components happens along the secretory pathway and at the plasma membrane, the...
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| Format: | Dissertation |
| Sprache: | Englisch |
| Veröffentlicht: |
Philipps-Universität Marburg
2019
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| Zusammenfassung: | Fungi, such as the unicellular model organism Saccharomyces cerevisiae, possess a thick cell wall composed of polysaccharides and proteins, which is essential for viable and healthy cells. While the mere synthesis of its components happens along the secretory pathway and at the plasma membrane, the correct processing is established on the exterior side of the plasma membrane. This is realized by a set of enzymes, called glycoside hydrolases (GH), which act on the hydrolysis and rearrangement of glycosidic bonds. In S. cerevisiae and the human pathogen Candida albicans, it has been shown that members of the GH76 family (Dfg5-subfamily) carry out the incorporation of GPI-anchored proteins into the cell wall, which is essential for these organisms. Although bacterial homologs of that class were already described, our understanding of the fungal counterparts and their underlying mechanism with its exceptional potential as a drug target still lack behind.
In order to fill this gap, the GH76 family has been subjected initially to phylogenetic analysis providing insights into its multifunctional character with up to ten different subfamilies. The exact role of Dfg5-proteins could be explained by an in-depth structural and functional analysis of one of its members, CtDfg5, from the thermophilic mold Chaetomium thermophilum. Its crystal structure determined at atomic resolution showed that the overall fold and the active site motif is shared between fungal and bacterial homologs, however an annotated function as α1,6-mannanases could not be shown in vitro. Instead it was possible to reassemble the GPI-core glycan structure (Manα1,2-Manα1,6-Manα1,4-GlcN) within the substrate-binding pocket of CtDfg5 by screening crystals with high molar sugar-fragments. This did not only provide a detailed view on the true substrate of Dfg5-proteins, but also first experimentally derived insights into the three-dimensional architecture of the GPI anchor glycan. Together with the complex structure of a putative acceptor molecule, a lipid-to-wall transfer mechanism catalyzed by Dfg5-proteins could be derived. Moreover, the structural insights suggested a possible way of using different GPI-modifications as a coding system to determine the final localization of GPI-anchored proteins. Furthermore, structure-based docking of a commercially available lead library helped to identify a small molecule (FP-1) that binds to the active site of CtDfg5. FP-1 shows specific effects at milli molar concentrations in terms of the viability of the model organism S. cerevisiae, assuming a high potential for further drug development. Finally, another fungal homolog from a so far uncharacterized subfamily showed that all GH76 family members recognize α1,6 mannobiose as a central element of cognate substrates. |
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| Umfang: | 1 Seiten |
| DOI: | 10.17192/z2019.0511 |