Function of PfNCR1 at the PPM/PVM Interface

Summary Niemann-Pick C1-related protein 1 (PfNCR1) is a cholesterol (CHL) transporter that is of great interest as an antimalarial drug target. PfNCR1 resides in regions of the parasite plasma membrane that are in contact with the parasitophorous vacuolar membrane that surrounds the malaria parasite inside its host erythrocyte. It is believed that this transporter defends the CHL-poor plasma membrane from CHL accumulation by pumping CHL out. We have recently obtained a cryo-EM structure of PfNCR1, alone and in complex with a small molecule inhibitor. The structures reveal a CHL tunnel that appears to be blocked by inhibitor. Other known inhibitors are predicted by molecular modeling to block CHL movement through the tunnel. We have identified two new PfNCR1 inhibitors that are predicted to bind away from the tunnel region and are putative allosteric inhibitors. We propose to characterize the mechanism of inhibition of PfNCR1 by these interesting new inhibitors, using structural, physiological, genetic and cell biological approaches. We recently described a region of contact between the parasite plasma membrane and parasitophorous vacuolar membrane in a study of PfNCR1 localization. We proposed that the contact sites could be where hydrophobic solutes are transported, in contrast to the regions where there is a substantial vacuolar space between the two membranes, wherein the protein export machinery resides. Little is known about the region of apposition. Our goal is to take advantage of the PfNCR1 localization to explore the components of this zone. Preliminary proximity biotinylation experiments have shown a number of transporters in proximity to PfNCR1. We propose to study these associations by protein biochemistry and structural studies. The goal is to develop a better understanding of components and interactions in this membrane contact zone, to inform us about function of the region. We anticipate that the proposed studies will give us new insights into an important cellular transporter, PfNCR1, its inhibition, and its function in the unexplored region of apposition between the two membranes that surround the malaria parasite. The proposed work is a terrific collaboration between the cell biology/biochemistry group of Dr. Daniel Goldberg and the structure/physiology group of Dr. Ed Yu. This collaboration is already underway and functioning beautifully. This grant aims to keep this work moving forward. Project Number: 1R01AI192669-01 | Fiscal Year: 2025 | NIH Institute/Center: National Institute of Allergy and Infectious Diseases (NIAID) | Principal Investigator: Daniel Goldberg (+1 co-PI) | Institution: WASHINGTON UNIVERSITY, SAINT LOUIS, MO | Award Amount: $633,218 | Activity Code: R01 | Study Section: Pathogenic Eukaryotes Study Section[PTHE] View on NIH RePORTER: https://reporter.nih.gov/project-details/1R01AI19266901