Structural and functional insights into KIDINS220-mediated regulation of the phosphate exporter XPR1

<title>Abstract</title> <p> XPR1 is the sole known protein that transports inorganic phosphate (Pi) out of cells in metazoans, dynamically regulated by inositol polyphosphates (InsP) signaling and protein interactions to maintain cellular phosphate homeostasis. While InsP-mediated regulation is well-characterized, the mechanistic role of direct protein interactors remains poorly understood. Here, we elucidate the structural and functional interplay between XPR1 and its regulatory partner KIDINS220 using cryo-EM, live-cell FLIM-FRET, and functional transport assays. We demonstrate that KIDINS220 stabilizes an unconventional conformation of the XPR1 SPX domain characterized by a ~ 180° rotation. This rearrangement establishes dual InsP ₆ -anchored interfaces, allosterically repositioning the SPX N-terminus toward the XPR1 C-terminal region and trapping the transporter in an inactive state. FLIM-FRET profiling visualizes and confirms that KIDINS220 promotes a more compact and conformationally homogeneous population of XPR1 in live cells. Under InsP ₆ -bound conditions, KIDINS220 enforces TM9b closure and cytoplasmic C-plug occlusion, obstructing the Pi permeation pathway. Upon Pi sensing, TM9b transitions to an open conformation, yet persistent C-plug engagement—mediated by hydrophobic and electrostatic networks—prevents substrate translocation. Functional validation demonstrates that KIDINS220 suppresses XPR1-mediated Pi transport, establishing its role as a dual-functional regulator: a trafficking chaperone and a membrane-localized brake. We propose a cooperative gating model where Pi acts as both chemical substrate and allosteric trigger to overcome KIDINS220-imposed inhibition. This work defines a tiered regulatory paradigm integrating protein scaffolding, ligand sensing, and dynamic gating for precision control of phosphate homeostasis. </p>