Abstract
Human SAMHD1 is a critical dNTP triphosphohydrolase. This enzyme is involved in regulating cellular concentrations of dNTPs, acts as an antiviral factor for retroviruses like HIV, and has been thought to be involved in innate cancer prevention. These functions highlight the promising nature of SAMHD1 as a drug target. The catalytic activity of Hs SAMHD1 is governed through a two-step tetramerization process, specific oligomerization patterns, and metal-ion binding. Although many studies have been conducted on Hs SAMHD1, little is known about its exact mechanisms. Our study focuses on elucidating mechanistic information about SAMHD1 from the Leishmania tarentolae ortholog, Lt SAMHD1, a parasitic SAMHD1. Not only does this ortholog provide a simpler template to investigate, but provides an antiparasitic therapeutic target due to the role of parasitic SAMHD1 in African trypanosomiasis, or “sleeping sickness.” In this study, we aim to uncover details about the mechanism of catalysis and allosteric activation of Lt SAMHD1 via various mutants. We found that like the human enzyme, Lt SAMHD1 conserves the histidine residue important for coordination of the second metal ion in the active site. Additionally, we confirmed that Lt SAMHD1 follows a similar activation pattern by tetramerization but prefers binding of GTP at the first allosteric site and dCTP at the second allosteric site. We also discovered that allosteric site 1 is imperative for protein stability; mutations at allosteric site 1 severely destabilize the protein and result in insolubility. These results provide the first fundamental basis towards our understanding of how parasitic SAMHD1s regulate their activity and select for their substrates. Our work has set the grounds for establishing the basic mechanism of dNTP regulation in parasitic SAMHD1s and provides a framework for the development of therapeutic avenues.