G protein-coupled receptors (GPCRs) are integral membranes that play a key role in fungi through sensing the extracellular environment. There are 10 distinct families of GPCRs in fungi, and they can bind a wide variety of molecules (agonists) including nutrients (e.g. sugars, amino acids) and peptides (e.g. a-factor, alpha-factor). Binding of agonists to the GPCR causes its activation, coupling to a G protein, nucleotide exchange and then activation of downstream signalling pathways that alters the cellular biochemistry. Yeast GPCRs have not been well-characterised structurally, and there is currently only a single GPCR structure, Ste2, from Saccharomyces cerevisiae that was determined recently by our group by cryo-EM. The aim of this PhD project is to determine one or more structures of GPCRs in other families, distinct from Ste2, to understand the diversity of signalling mechanisms in fungi. We will prioritise GPCRs that are found in pathogenic fungi, which may have the potential to act as drug targets. Currently, treatments for systemic fungi infections in immune compromised patients are limited and ineffective, resulting in high mortality rates.
The project will start with cloning a number of GPCRs from different pathogenic fungi, expressing them using recombinant baculovirus in insect cells, purification, formation of G protein-coupled complexes and structure determination by cryo-EM. The successful candidate will be expected to develop and master all the steps of the procedure and to become competent in computational approaches of image processing, structure determination and model building. In addition, other biophysical techniques will be used (e.g. FSEC, SEC-MALs, SPR) in combination with a suite of biochemical assays to characterise the purified proteins and their cellular function.
References
Velazhahan, V., Ma, N., Vaidehi, N. & Tate, C.G. (2022) Activation mechanism of the class D fungal GPCR dimer Ste2. Nature 603: 743-748.
Velazhahan, V., Ma, N., Pándy-Szekeres, G., Kooistra, A.J., Lee, Y., Gloriam, D.E., Vaidehi, N. & Tate, C.G. (2021) Dimeric structure of the Class D GPCR Ste2 coupled to two G proteins. Nature 589: 148-153.