Quinone Reductase 2 Dimerization is Dynamically Driven by Ligand Binding

Abstract Human quinone reductase 2 is a cytosolic flavoprotein involved in cell physiology and metabolism, and implicated in several diseases. However, the mechanisms that govern its oligomeric assembly and diverse functional outcomes remain incompletely understood. Here, we employ native mass spectrometry to directly resolve the dynamic oligomeric landscape of recombinant human quinone reductase 2 expressed in Escherichia coli, preserving noncovalent interactions and enabling analysis of assembly behavior under native conditions. Quinone reductase 2 is predominantly observed as a dimer stabilized by multiple noncovalently bound ligands, giving rise to discrete species. Top-down native mass spectrometry reveals a single intact proteoform, excluding covalent modification or covalently bound flavins as drivers of oligomerization. Binding of flavin adenine dinucleotide robustly stabilizes the dimer, while unexpectedly, flavin mononucleotide also promotes dimer formation. As flavin mononucleotide and flavin adenine dinucleotide differ structurally by the presence of an adenine dinucleotide moiety, we hypothesized that purine nucleotide binding itself may modulate quinone reductase 2 assembly. Consistent with this, we identify a new concentration-dependent effect of guanosine triphosphate on quinone reductase 2 dimerization. Functional reductase assays show that flavin-stabilized dimers exhibit the highest catalytic activity, whereas guanosine triphosphate-induced dimers retain reduced activity. Binding of the inhibitor YB537 abolishes activity despite promoting dimer formation. Together, these findings reveal ligand-dependent structural plasticity in quinone reductase 2 oligomerization that is decoupled from reductase activity. This supports a model in which dimerization is shaped by the local ligand environment, enabling distinct functional states and suggesting a broader regulatory role beyond reductase catalysis.