The cancer-promoting enzyme PKM2 binds RNA via a positively charged regulatory patch

ABSTRACT: Pyruvate kinase M2 (PKM2) is a glycolytic enzyme that coordinates energy production with biosynthetic demands in physiologically proliferating and cancer cells. PKM2 has also emerged as a non-canonical RNA-binding protein. Here, we combine targeted PKM2 mutagenesis with RNA-protein interaction assays, biochemical and biophysical analyses to define how RNA binding relates to PKM2 allosteric control and oligomeric state. We show that the allosteric activator fructose-1,6-bisphosphate (FBP) strongly reduces PKM2 binding to RNA. Mutants with impaired oligomerization show reduced RNA binding, and RNA association is favored by the tetrameric state. A positively charged surface patch in the FBP-binding region is essential for RNA binding and displays emergent properties. Single-residue variants further link RNA association to FBP-responsive tetrameric conformations. Our data integrate RNA binding with PKM2 allosteric regulation.