Structure and mechanism of tRNA splicing enzymes Trl1 and Tpt1: therapeutic targets for fungal infection.

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Invasive fungal infections, especially Aspergillosis and Candidiasis, are a major cause of morbidity and mortality in cancer patients with prolonged neutropenia following chemotherapy or hematopoietic stem cell transplantation. The development of more effective (and less toxic) drugs for the treatment of fungal infections in cancer settings hinges on defining new targets for antifungal drug discovery and implementing strategies to identify inhibitors. Although the tRNA splicing enzymes are promising anti-fungal drug targets, a prime bottleneck is the lack of atomic structures for the unique fungal tRNA ligase (Trl1) and 2´-phosphotransferase (Tpt1). To that end, I intend to pursue structural, biochemical and genetic characterization of the Trl1 ligase domain and Tpt1 from the fungal pathogens Aspergillus fumigatus and Candida albicans. The premise of the project is that clarifying the structures and mechanisms of the Aspergillus and Candida tRNA splicing enzymes will drive progress toward drug discovery. The specific aims for the 2-year project proposal are: 1. To crystallize and determine the atomic structures of the Trl1- ligase domain of fungal pathogens, in complexes with substrates, cofactors, and reaction intermediates. 2. To determine crystal structures of Tpt1 in complexes with substrates (NAD+ and a synthetic RNA with a 3´-5´-phosphodiester, 2´-phosphate splice junction) and to trap Tpt1 in a complex with the RNA 2´- phospho-ADP-ribosylated reaction intermediate.This will fill a major knowledge gap in tRNA metabolism by providing insights to involved active site architectures and catalytic mechanisms. The structure-function information will promote the discovery of antifungals.