A mechanism for cancer-associated inactivation of NQO1 due to P187S and its reactivation by the consensus mutation H80R.

The cancer-associated P187S polymorphism in the NAD(P)H:quinone oxidoreductase 1 (NQO1) abolishes enzyme activity by strongly reducing FAD binding affinity. A single mammalian consensus mutation (H80R) protects P187S from inactivation. To provide mechanistic insight into these effects, we report here a detailed structural and thermodynamic characterization of FAD binding to these NQO1 variants. Our results show that H80R causes a population shift in the conformational ensemble of apo-P187S, remodelling the structure and dynamics of the FAD-binding site and reducing the energetic penalization arising from the equilibrium between apo- and holo-states. Our analyses illustrate how single amino acid changes can profoundly affect structural and mechanistic features of protein functional traits, with implications for our understanding of protein evolution and human disease.