NMR spectroscopy studies to elucidate the role of SPX domains in phosphate homeostasis

Pipercevic, Joka. NMR spectroscopy studies to elucidate the role of SPX domains in phosphate homeostasis. 2021, Doctoral Thesis, University of Basel, Faculty of Science.


Official URL: https://edoc.unibas.ch/85405/

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This PhD thesis focuses on two main areas of research. The first area deals with structural and functional studies of SPX domains in yeast and plants that are key to the understanding of the basis of eukaryotic phosphate (Pi) homeostasis. A better understanding potentially offers a door-opener for future treatment of human primary familial brain calcification disease. Furthermore, engineered highly improved crops with better phosphate efficiencies could help to fight the imminent crisis due to overuse of soil biodiversity destructive Pi fertilizers and the limited world supply in Pi rock.
The second part deals with the pore-forming proteins bacterial Escherichia coli Colicin Ia and human Gasdermin D. Working with pore-forming toxin Colicin Ia can pave the way for novel drug-like antibiotics targeting E. coli related strains. The structure and function of Gasdermin D can help to develop therapeutics targeting aberrant immflammatory response causing neurodegenerative diseases like Alzheimer’s, autoimmflamatory disorders e.g. multiple sclerosis and macular degeneration as well as cardiometabolic disease type 2 diabetes.
This work focuses on SPX domains from yeast Saccharomyces cerevisiae. SPX domains are present in many functionally different proteins linked to Pi homeostasis. For the first time we were able to structurally and dynamically characterize a SPX domain in solution state by NMR discovering a previously unknown α-helix7 expanding the known SPX domain boundaries. This α-helix7 most likely plays a key role to diversify protein function of SPX-containing proteins. The molecular mechanism of the SPX-containing VTC complex in dependence of inositol pyrophosphates that are known to modulate Pi homeostasis was elucidated. We show for the first time that SPX domains form of a non- active inhibitory state comprised of a SPX-SPX heterodimer. Upon addition of inositol pyrophosphates or inositol phosphate this interaction is disrupted forming single monomers. The ligand binding induces a conformational change of the conserved α- helix1 reorienting. The α-helix1 is not only involved in the ligand binding, known from earlier studies, but also in protein-protein interactions. α-helix1 contributes to the binding to other SPX domains as well as to the central TTM domain of Vtc2 suggesting a competitive binding mode. We propose a molecular mechanism for the function of SPX to consist of two main events – the inhibition of the function of SPX domain in the absence of inositol pyrophosphates and a second event activating the function of SPX 2
protein by inositol pyrophosphates, an event that is most likely protein specific. For SPX system in VTC complex, we speculate that the α-helix7 could get exposed upon inositol (pyro-) phosphate binding and subsequently phosphorylated by cellular kinases and pyrophosphorylated by inositol pyrophosphates enabling the activation of ATPase activity for the required synthesis of polyphosphate chains. The findings of this study offer a new dimension for understanding cellular Pi homeostasis regulation.
My work on the role of SPX domains in plants resulted in a co-authored paper that can be found in the attachments. Here we show that the coiled-coil (CC) domain of a PSR transcription factor - known to interact with a stand-alone SPX protein in the presence of inositol pyrophosphates - is able to bind inositol pyrophosphate by itself. This is suggesting inositol pyrophosphate to act as molecular glue between stand-alone SPX proteins and the PSR transcription factor. This complex is important for inactivation of Pi-starvation genes under conditions with excess Pi.
Furthermore, I performed electron microscopy and x-ray crystallography studies on membrane-bound Colicin Ia demonstrating that the only known structure of colicin - the membrane-inserted pore of Colicin Ia at a low-resolution by negative-stain electron microscopy - is actually a contamination. We identified the contamination to be the oligomeric protein Dps form E. coli induced under mitomycin C reagent. The manuscript is in the submission phase and attached to this thesis. It will help the scientific community to rethink potential pore-forming mechanisms based on this wrong interpretation of the data.
Finally, the cryo-electron microscopy I conducted on caspase-cleaved Gasdermin D shed light on its previously unclear function. Gasdermin D was known to be a crucial substrate for inflammatory caspases causing pyroptosis via IL-1β and IL-18, however, the mechanism of Gasdermin D was still unknown. I observed gigantic membrane pores in the liposomes that were not reported before identifying the Gasdermin D as a pore- forming protein.
Advisors:Hiller, Sebastian and Basler , Marek
Faculties and Departments:05 Faculty of Science > Departement Biozentrum > Structural Biology & Biophysics > Structural Biology (Hiller)
UniBasel Contributors:Basler, Marek
Item Type:Thesis
Thesis Subtype:Doctoral Thesis
Thesis no:14575
Thesis status:Complete
Number of Pages:108
Identification Number:
  • urn: urn:nbn:ch:bel-bau-diss145757
edoc DOI:
Last Modified:15 Feb 2022 11:01
Deposited On:18 Jan 2022 09:21

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