Structural basis for substrate recognition and inhibition of human glucose-6-phosphate transporter SLC37A4.

Glucose 6 phosphate (G6P) homeostasis is essential for maintaining blood glucose levels and coordinating anabolic and catabolic pathways. A key step in this process is the delivery of G6P into the endoplasmic reticulum (ER), where it is hydrolyzed by glucose 6 phosphatase to glucose and inorganic phosphate (Pi). This transport step is carried out by the ER carrier SLC37A4 (also known as the G6P transporter, G6PT), which imports G6P into the ER lumen while exporting Pi to the cytosol, and loss-of-function mutations in SLC37A4 cause glycogen storage disease type Ib. Despite its central role in G6P homeostasis, how SLC37A4 recognizes G6P and couples its transport to Pi antiport has remained unclear. Here we report cryo-electron microscopy structures of human SLC37A4 in three states: the apo form at 2.8 Å resolution, a G6P-bound state at 3.2 Å resolution and a chlorogenic acid (CHA) bound state at 3.3 Å resolution. SLC37A4 adopts the canonical Major Facilitator Superfamily fold and harbors a central, positively charged cavity that accommodates anionic substrates. In the G6P-bound structure, SLC37A4 adopts an outward-open conformation facing the ER lumen, in which G6P binds to the electropositive pocket. In the CHA-bound structure, SLC37A4 adopts an inward-facing conformation, with CHA bound at a cytosolic site that locks the transporter in an arrested state and prevents the conformational transitions required for G6P/Pi exchange. Combined with thermostability and transport-based analyses of G6P binding and disease variants, these structures support a rocker switch mechanism in which electrostatic neutralization of the central positively charged cavity by anionic substrate drives the conformational changes underlying G6P/Pi exchange. Together, these findings define the structural basis of G6P/Pi exchange by SLC37A4, provide a molecular rationale for pathogenic mutations in glycogen storage disease type Ib, and provide a framework for targeting SLC37A4 to modulate G6P homeostasis.