Identification of CDP-DAG and serine binding sites in Candida albicans phosphatidylserine synthase, an antifungal drug target

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Candida albicans is among the most common causes of fungal infections in humans. Candida causes oral infections in immunocompromised people such as those with AIDS, and it can cause even more serious invasive bloodstream infections in neutropenic patients such as those undergoing cancer chemotherapy and organ transplants. Although there are three classes of antifungals to treat invasive fungal infections, a combination of drug resistance and toxicity limit their effectiveness. Thus, there is a need for new classes of antifungals. The Cho1 phosphatidylserine (PS) synthase represents a potential new drug target for three reasons: 1) Loss of PS synthase blocks virulence, and causes clearance of the fungus in mouse models of systemic or oral infection. 2) PS synthase is not homologous to mammalian (i.e. human) PS synthases, which use a different mechanism to synthesize PS. Mammalian PS synthases exchange head groups from phosphatidylethanolamine (PE) or phosphatidylcholine (PC) for serine to make PS. Fungal PS synthase condenses cytidyldiphosphate-diacylglycerol (CDP-DAG) and serine to make PS. 3) Fungal PS synthases are conserved in all fungal pathogens, so PS synthase inhibitors could be broad spectrum. One approach to developing enzyme inhibitors is to look for competitive inhibitors of the enzyme's natural substrates. This approach would be enhanced by a more comprehensive understanding of Cho1's interactions with its substrates: CDP-DAG and serine. Elucidation of the binding motifs for both substrates would allow us to better design potential competitive inhibitors, such as serine analogs, to act as lead compounds for antifungal development. A common CDP-binding consensus motif that is present in CDP-alcohol phosphotransferases is [D-(X)2-D-G-(X)2-A-R-(X)8-G-(X)3-D-(X)3-D], and this is found in Cho1. However, the serine-binding motif for Cho1 is unknown. Using homology sequence alignments and computational tools that superimpose Cho1 on the solved structure of a prokaryotic CDP-alcohol phosphotransferase, a putative serine binding motif was identified. Two aims will be pursued to confirm the CDP-DAG binding motif and discover the serine-binding motif. Aim 1. Genetic analysis will be used to identify residues important for substrate binding in Cho1. Based on the known CDP binding motif, alanine-scanning mutagenesis will be used to test the importance of this motif in PS synthesis. Alanine-scanning mutagenesis will also be used to test the function of the putative serine binding motif. Aim 2: Chemical cross-linking with serine analogs will be used to identify the serine binding domain. In a less-biased biochemical approach, serine analogs have been synthesized that compete with native L-serine for PS synthesis. This set of analogs will be further developed and used to cross-link the substrates to the PS synthase, and follow this with mass spectrometry to identify amino acids with which they interact. Interacting amino acids will then be genetically analyzed using alanine-scanning mutagenesis.