Lin, Y.-C.; Luo, S.; Patil, A.; Primanis-Erickson, N.; Jiang, X.; Webb, B.; Hsu, K.-L.

Phosphofructokinase-1 (PFK-1) catalyzes the ATP-dependent conversion of fructose-6-phosphate (F6P) to fructose-1,6-bisphosphate (F1,6BP), the first committed step of glycolysis. Beyond classical allostery, the liver isoform PFKL forms higher-order assemblies, but how ligand binding redirects intermolecular interactions remains unclear. Here we use high-speed atomic force microscopy (HS-AFM), topology-based AFM image simulations, and molecular dynamics (MD) simulations to define ligand-dependent assembly switching of human PFKL. Wild-type PFKL (PFKL WT) forms lattice-like assemblies under APO and ATP conditions, whereas coordinated ATP and F6P loading redirects assembly toward filaments in a ligand-order-dependent manner. APO-PFKL WT also forms lattice-like assemblies on or near membrane-supported surfaces, suggesting that interfacial environments influence where assembly initiates. The filament-defective N702T mutant forms ordered double-layer lattices and preserves this geometry under APO, ATP, and ATP+F6P conditions. MD simulations suggest that lattice stabilization arises from distributed inter-tetramer contacts. These findings define a structural framework for ligand-dependent supramolecular regulation of glycolysis.