Excellence in Research: Determination of Structure and Substrate-Specificity of a Unique HAD Phosphatase in Mitochondria

Mitochondria play a vital role in many cellular functions, including metabolic regulation, signal transduction, and controlled cell death. Mitochondria require hundreds of proteins to do these functions; however, more than ninety-five percent of mitochondrial proteins are encoded in the nuclear DNA. Therefore, these proteins are imported into the mitochondria after synthesis in the cytosol via translocase of the mitochondrial outer and inner membranes referred to as the Tom and Tim proteins, respectively. In addition to mitochondrial protein translocation, the Tom and Tim proteins often perform additional functions via their intrinsic enzymatic properties and play a broader role in cellular health. The Tim50 homolog in a group of ancient parasitic protozoa possesses the haloacid dehalogenase (HAD) phosphatase activity but the significance of this enzymatic activity is not clear. This project will utilize different structure biology tools to characterize the structure and elucidate the dual roles of a mitochondrial HAD-phosphatase in a divergent unicellular organism. The project will provide hands-on research experiences for graduate/undergraduate minority students from local colleges and universities in various biochemical and structural biology works. The outcome for trainees will be their ability to combine structural and biological approaches to address broad questions at the interface of biology and physical chemistry. Tim50 is an essential protein in eukaryotes. The HAD signature motif is specifically found in the protozoal Tim50. In humans, Tim50 has protein phosphatase activity and is involved in wider cellular functions. It is over-expressed in many types of cancer cells. Inherent mutations of Tim50 are connected to severe neurological disorders. As the receptor translocase, the C-terminal domain of Tim50 binds with signal peptides of various mitochondria-targeted proteins. Interestingly, the same domain in Tim50 is also involved in binding phospho-substrates. As the structural information is very limited, the mechanism of action for the dual function of Tim50 is elusive. This project is focused on characterizing the structure, membrane topology, substrate specificity, and other biochemical characteristics of the protozoal Tim50 to understand the significance of its dual function, which can be extrapolated to Tim50s in other systems and elucidate the evolutionary perspective of Tim50. This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.