This proposal seeks to understand how proteins located in the cell membrane work as gatekeepers to selectively allow compounds into or out of the cell. Such gatekeepers are known as or ATP-binding cassette (ABC) transporters, because they use the energy of ATP (adenosine triphosphate) hydrolysis to transport compounds across the cell membrane. Bacterial ABC importers are essential for organism survival, controlling the rate of uptake for nutrients scavenged from the bacterium's environment. Control of the rate of transport precludes over-accumulation of a nutrient that is beneficial at low concentrations, but is potentially toxic at high concentrations. While a subset of ABC proteins contain an additional ?accessory? domain that can regulate the uptake of compounds by shutting off the transporter, it is unclear why certain transporters contain these domains while others do not. However, we do understand that certain transporters are ?turned off? when a specific compound or protein binds to this accessory domain. Other accessory domains regulate by ?sensing? changes in the microenvironment and reacting accordingly. To decipher this mechanism of regulation, the PI's laboratory combines biochemical and biophysical experiments with structural biology to understand how these accessory domains play a role in transport regulation, which in restricts or allows nutrients to enter the cell. This research program will define the molecular mechanism that controls nutrient uptake and allow researchers to understand how multiple transport systems work in concert within an organism to maintain cell survival. We will test our hypothesis that regulation of transporter activation via a sensing accessory protein. The proposed research will decipher the complex circuitry of regulation in a model system in three Aims to: (1) understand how PepT SBPs select for different substrates within the microenvironment (i.e., nutrients, cofactors and peptides); (2) determine how the assembly of the core transporter dictates transport selectivity and efficiency (3) reveal how PepT transporters regulate the import of substrates into the cell through the activation of a novel regulatory domain. This research program has set out to close critical gaps in the understanding of the fundamentals of the transport mechanism present in all bacteria. The results will yield insights into how regulatory domains modulate transport across all organisms, crucial for cell viability.
ATP-binding cassette (ABC) transporters utilize many mechanisms to ensure efficient nutrient uptake while regulating toxins. Regulatory domains are not conserved in all ABC importers, indicating more variability in ABC importers beyond the current Type I/II classification scheme (Rice, Park et al., 2014). The proposed research will define the mechanism in which transporters can shut off transport based on environmental cues, which is a critical, but often overlooked, aspect to understanding basic nutrient uptake and survival mechanisms.
