The Chemical Biology and Bioinorganic Chemistry of Yeast Phosphatidylinositol Transfer Protein

Phosphatidylinositol Transfer Proteins are an enigmatic family of proteins present across the plant and animal kingdoms. They act as diversifiers of phosphoinositide signaling and regulate diverse cellular processes such as membrane trafficking in yeast, polarized root hair growth in plants and neural stem cell development in mouse. Model organism Saccharomyces cerevisiae has provided opportunities to study Sec14, a prototype PITP, in detail. Sec14 orchestrates a kinase dependent phosphoinositide signaling pathway in cells that co-ordinates phosphatidylcholine metabolism with transGolgi network/endosomal dynamics. It is essential to the infectivity of many fungal pathogens and is thus an attractive drug target. Small molecule inhibitors belonging to nitrophenyl piperazin methanone (NPPM) scaffold have been developed but their mode of action is not clear. Using genetic screens and in vitro reconstitutions of phosphatidylinositol transfer activity, I studied the mechanism by which NPPMs inhibit Sec14. I identified a motif associated with Sec14 that allows pan-fungal predictions of sensitivity to NPPMs. My work showed that Sec14 from fungal pathogen Candida glabrata is sensitive to NPPMs. Two new classes of Sec14 inhibitors belonging to picolinamide and benzamide scaffolds, were identified and rigorously validated. These small molecules are potential lead compounds for novel anti-fungal drug development. I also studied Sfh5, a Sec14 homolog from yeast. I discovered that Sfh5 is a heme binding protein. Using Electron Paramagnetic Resonance and Mössbauer spectroscopy, I determined the biophysical properties of the heme iron center. This is the first known instance of a PITP that binds heme. The structure of Sfh5 was solved at 2.7Å resolution through X-Ray crystallography, revealing a novel mode of heme binding. We also identified specific mutations in Sfh5 that weakened or abrogated heme binding. My studies reveal an allosteric relationship between phosphatidylinositol and heme binding sites of the protein. Finally, I show that when Sfh5 is unable to bind heme, it acquires robust phosphatidylinositol transfer and signaling abilities. These studies open avenues for development of a new class of anti-fungal drugs and offer new insights into the structure and functions of PITPs