Human NAD(P)H:quinone oxidoreductase 1 (NQO1) is a multi-functional protein whose alteration is associated with cancer, Parkinson's and Alzheimer´s diseases. NQO1 displays a remarkable functional chemistry, capable of binding different functional ligands that modulate its activity, stability and interaction with proteins and nucleic acids. Our understanding of this functional chemistry is limited by the difficulty of obtaining structural and dynamic information on many of these states. Herein, we have used hydrogen/deuterium exchange monitored by mass spectrometry (HDXMS) to investigate the structural dynamics of NQO1 in three ligation states: without ligands (NQO1apo), with FAD (NQO1holo) and with FAD and the inhibitor dicoumarol (NQO1dic). We show that NQO1apo has a minimally stable folded core holding the protein dimer, with FAD and dicoumarol binding sites populating binding non-competent conformations. Binding of FAD significantly decreases protein dynamics and stabilizes the FAD and dicoumarol binding sites as well as the monomer:monomer interface. Dicoumarol binding further stabilizes all three functional sites, a result not previously anticipated by available crystallographic models. Our work provides an experimental perspective into the communication of stability effects through the NQO1 dimer, which is valuable for understanding at the molecular level the effects of disease-associated variants, post-translational modifications and ligand binding cooperativity in NQO1.

Center for Rare Diseases Hospital Universitario de Canarias Universidad de La Laguna 38320 Tenerife Spain

Department of Biochemistry Faculty of Science Charles University Hlavova 2030 8 128 43 Prague 2 Czech Republic

Department of Physical Chemistry and Unit of Excellence in Chemistry University of Granada Av Fuentenueva s n E 18071 Granada Spain

Institute of Microbiology Academy of Sciences of the Czech Republic Videnska 1083 142 20 Prague 4 Czech Republic

School of Pharmacy and Biomolecular Sciences University of Brighton Huxley Building Lewes Road Brighton BN2 4GJ UK

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