Structure and ligand binding of receptor adenylate cyclase from Trypanosoma brucei

/ ABSTRACT Trypanosoma brucei is a flagellated eukaryotic pathogen responsible for African trypanosomiasis, a vector- borne disease that causes enormous human suffering and economic burden in sub-Saharan Africa, where it is endemic. In addition to its medical and agricultural relevance, T. brucei is a scientifically interesting study organism, which serves as a model for related human parasites and early branched eukaryotes. Moreover, as a single-celled organism with easy genetic manipulation, T. brucei is also valuable for studies of flagellum (aka cilium) biology. In order to be transmitted, survive, and cause disease, T. brucei must sense and respond to environmental signals. This is particularly important for a vector-transmitted parasite because it must adapt to diverse vector and host environments. Signaling is achieved by signal transduction pathways that detect environmental signals, transduce them into an intracellular signal (second messenger), and trigger a cellular response. However, very little is known about these signaling pathways, in T. brucei and related pathogens, presenting a critical knowledge gap and window of opportunity for the identification of species-specific drug targets. Cumulative evidence from our lab and others has shown that the T. brucei flagellum is a cAMP signaling platform, controlling different aspects of T. brucei biology, transmission, and pathogenesis. For instance, movement, cell-cell communication, chemotaxis, movement through tissues, transmission thorough the insect vector, and pathogenesis in the mammalian host, are all influenced by flagellar cAMP signaling systems. cAMP signaling initiates with receptor-type adenylate cyclases (ACs) on the flagellar membrane. T. brucei ACs are transmembrane proteins with an intracellular catalytic domain, responsible for production of cAMP, connected by a single-pass transmembrane domain two extracellular Periplasmic Binding Protein (PBP) domains, presumably responsible for ligand binding and regulation. In bacteria, PBPs bind to small molecules around the size of an amino acid, and control a variety of biological phenomena, including internalization of bound ligands and chemotaxis. Surprisingly, despite their vital role in initiating cAMP signal transduction, the structure of T. brucei AC PBP domains and identity of putative ligands have not been experimentally determined, and this is central goal of the present project. Our specific aims are to (1) determine the 3D structure of AC1 and (2) test candidate ligand binding to AC1. To determine the 3D structure of AC1, we will purify native, HA-tagged AC1 directly from T. brucei for atomic-resolution structure determination by cryoEM. This aim will provide insights into ligand binding sites in AC1, shortening the list of candidates to be tested in aim 2. To test candidate ligands, we will employ a heterologous expression system for AC1 expression in HEK cells, which then will be used to assess binding of candidate ligands directly using isothermal titration calorimetry. The proposed project will greatly expand our knowledge of environmental sensing by medically and economically important protozoan pathogens. Project Number: 1R03AI182978-01A1 | Fiscal Year: 2026 | NIH Institute/Center: National Institute of Allergy and Infectious Diseases (NIAID) | Principal Investigator: KENT HILL | Institution: UNIVERSITY OF CALIFORNIA LOS ANGELES, LOS ANGELES, CA | Award Amount: $157,500 | Activity Code: R03 | Study Section: Macromolecular Structure and Function C Study Section[MSFC] View on NIH RePORTER: https://reporter.nih.gov/project-details/1R03AI18297801A1