Biophysical and functional perturbation analyses at cancer-associated P187 and K240 sites of the multifunctional NADP(H):quinone oxidoreductase 1.

Once whole-genome sequencing has reached the clinical practice, a main challenge ahead is the high-throughput and accurate prediction of the pathogenicity of genetic variants. However, current prediction tools do not consider explicitly a well-known property of disease-causing mutations: their ability to affect multiple functional sites distant in the protein structure. Here we carried out an extensive biophysical characterization of fourteen mutant variants at two cancer-associated sites of the enzyme NQO1, a paradigm of multi-functional protein. We showed that the magnitude of destabilizing effects, their molecular origins (structural vs. dynamic) and their efficient propagation through the protein structure gradually led to functional perturbations at different sites. Modulation of these structural perturbations also led to switches between molecular phenotypes. Our work supports that experimental and computational perturbation analyses would improve our understanding of the molecular basis of many loss-of-function genetic diseases as well as our ability to accurately predict the pathogenicity of genetic variants in a high-throughput fashion.