“Where there’s life, there’s phosphorus.”Phosphorus is one of the five fundamental elements of life, as essential as carbon, oxygen, hydrogen, and nitrogen. In living organisms, organic phosphorus primarily exists in the form of nucleic acids, phospholipids, and energy molecules, while inorganic phosphorus is found as hydroxyapatite in bones, free phosphate ions, or pyrophosphate. Among these forms, free phosphate ions are the most prominent circulating form of phosphorus. Phosphorus is both a boon and a bane for living organisms: while essential for structural, energy, and signaling molecules, excessive phosphate concentrations can interfere with biochemical reactions and form precipitates with multivalent metal ions like calcium and iron, posing health risks such as primary basal ganglia calcification (PBGC) or thrombosis. In PBGC patients, unmetabolized phosphate accumulates in neural tissue, forming calcium phosphate precipitates that lead to degenerative changes. Additionally, in blood coagulation, polyphosphates activate thrombin and coagulation factors, making excess phosphate a potential cause of thrombotic disorders. Maintaining cellular phosphate homeostasis, including timely "release" when necessary, is therefore a vital aspect of health.

To regulate phosphate levels, cells have evolved various transport proteins on their membranes for phosphate uptake and release. In higher organisms, particularly humans, while several inward phosphate transporters have been identified, only one molecular system capable of outward phosphate efflux has been discovered: the XPR1 protein. Encoded by the SLC53A1 gene, XPR1 releases phosphate in response to intracellular overload, regulated by inositol pyrophosphate. These molecules are produced in large quantities when intracellular phosphate levels rise abnormally, acting as signaling molecules for phosphate imbalance. XPR1 dysfunction leads to intracellular phosphate accumulation, affecting cell functions (e.g., osteoclast impairment) and causing phosphate precipitation and calcification in various tissues and organs. Evidence suggests that XPR1 mutations impair astrocytes' ability to release phosphate into the blood, leading to phosphate buildup in cerebrospinal fluid and calcium phosphate precipitation, resulting in pathological changes.

Despite its importance in maintaining phosphate homeostasis, the lack of structural information on XPR1 has hindered understanding of its molecular mechanism, particularly how it is activated by inositol pyrophosphate to mediate phosphate release.

The new research findings published by Dr. Cao Yu's team show that XPR1 is a biological macromolecular machine composed of two parts: a phosphate channel responsible for phosphate transport and a signaling receptor responsible for inositol pyrophosphate recognition. The transmembrane domain (TMD), consisting of ten transmembrane helices, forms a pore through which phosphate molecules can cross the cell membrane. This pore is gated by tryptophan 573, and it opens or closes as tryptophan 573 rotates. The cytosolic SPX domain forms the inositol pyrophosphate receptor structure, binding inositol pyrophosphate and controlling conformational changes in the TMD. However, unexpectedly, the study reveals that XPR1 contains two binding sites for inositol pyrophosphate. The first binding site, located between the SPX receptors, can bind either inositol pyrophosphate or inositol phosphate. Binding of these molecules induces dimerization of the SPX domains, stabilizing the TMD conformation and making it easier to open. The second binding site is located between the SPX and TMD domains. This site is characterized by a large spatial cavity and positively charged residues that are spaced far apart, making it difficult to capture smaller inositol phosphate molecules. Only the larger inositol pyrophosphate, due to its pyrophosphorylation, can fit into this "vacant" pocket and bind effectively. The discovery of this second, inositol pyrophosphate-preferring binding site resolves a previous scientific question about why XPR1 is more sensitive to inositol pyrophosphate than to inositol phosphate and more readily activated by it.

In addition to elucidating the structural basis of XPR1’s recognition of inositol pyrophosphate, this study also employs electrophysiological methods to demonstrate, for the first time, that XPR1 functions as a phosphate channel, rather than the previously assumed transporter protein. Combining structural biology and functional biology, the paper proposes that XPR1 is an inositol pyrophosphate-gated phosphate channel, acting as a dual-function molecular machine that enables cells to sense phosphate metabolic signals and respond by releasing phosphate. This research provides a structural foundation for understanding the cellular mechanisms underlying phosphate metabolism and homeostasis, and offers valuable insights for exploring the molecular pathology of related calcification diseases.

Dr. Cao Yu, researcher at Shanghai Precision Medicine Institute and Orthopedics Department of Shanghai Ninth People's Hospital, and Dr. Yu Ye from China Pharmaceutical University are the co-corresponding authors of this paper. The co-first authors include Lu Yi, a doctoral student at the Shanghai Precision Medicine Institute, Yue Chenxi, a doctoral student at China Pharmaceutical University, Dr. Zhang Li from the University of Texas Southwestern Medical Center, and Associate Researcher Yao Deqiang from Renji Hospital, Shanghai Jiao Tong University School of Medicine. The cryo-EM data collection and processing analysis for this study were carried out at the Electron Microscopy Center of Shanghai Precision Medicine Institute. The functional experiments were primarily conducted at the Bioimaging Platform, Protein Platform, and Omics Platform of Shanghai Precision Medicine Institute. This research was supported by the National Natural Science Foundation of China, the Shanghai Municipal Education Commission "Category IV Peak" Project, and the Innovation Team of High-Level Local Universities in Shanghai.