Structural insights into the mechanism of phosphate recognition and transport by human XPR1

XPR1 is the only known protein that transports inorganic phosphate (Pi) out of cells, and the function is conserved across species from yeast to mammals ^1-4 . Human XPR1 variants lead to cerebral calcium-phosphate deposition, which are associated with a neurodegenerative disorder known as primary familial brain calcification (PFBC) ⁵ . Here, we present the Cryo-EM structure of human XPR1 bound to Pi ions. XPR1 contains 10 transmembrane α-helices, forming an ion channel-like architecture that recognizes and transports Pi ions. Two arginine residues, subject to pathogenic mutation in PFBC families, line the translocation channel and serve to bind Pi ions. Clinically linked mutations of these arginine residues impair the Pi transport activity of XPR1. To track the movement of Pi ions within the translocation channel, we capture a mutant XPR1 in an alternative conformation. It reveals a rearrangement of intrahelical hydrogen bonds between a channel-lining tryptophan and two Pi-binding residues. This rearrangement links Pi recognition and transport, by means of flipping the tryptophan residue to propel Pi through the translocation channel. Our results provide mechanistic understanding of how XPR1 recognizes and transports phosphate ions across cell membrane, and they establish a framework for interpreting disease-related mutations and for the development of future therapeutics.