A Dynamic Core in Human NQO1 Controls the Functional and Stability Effects of Ligand Binding and Their Communication across the Enzyme Dimer

Abstract

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.

The Catalytic Cycle of the Antioxidant and Cancer-Associated Human NQO1 Enzyme: Hydride Transfer, Conformational Dynamics and Functional Cooperativity

“…The use in these studies of Dic, a potent competitive inhibitor of NAD(P)H [6,39], has supported the proposal that NAD(P)H binding may cause minimal structural changes [46,47] but affects the stability and structural dynamics of the active site, which contributes to the enhancement of catalysis by reducing the reaction free energy barrier(s) and/or promoting quantum tunneling effects [42,45] ( Figure 1B). Interestingly, Dic binding might also allow for the communication of local stability effects between active sites during the catalytic cycle [42,48,49]. The existence of functional and structural non-equivalence between the active sites may also explain the apparent negative cooperativity found for Dic binding (mainly reflected by inhibition studies with the holo-protein) [48,49].…”

“…NQO1 activity is also required for the activation of cancer Importantly, most of our knowledge on the NQO1 kinetic mechanism has come from analysis of either ligand binding/release events from crystallographic analyses, or from single-wavelength kinetic analyses focused on the changes in FAD spectral properties associated with chemical steps (i.e., HT) [34,38,40,43,44]. Several studies have supported that changes in molecular dynamics throughout the catalytic cycle should also be considered [42,45]. The use in these studies of Dic, a potent competitive inhibitor of NAD(P)H [6,39], has supported the proposal that NAD(P)H binding may cause minimal structural changes [46,47] but affects the stability and structural dynamics of the active site, which contributes to the enhancement of catalysis by reducing the reaction free energy barrier(s) and/or promoting quantum tunneling effects [42,45] ( Figure 1B).…”

“…Several studies have supported that changes in molecular dynamics throughout the catalytic cycle should also be considered [42,45]. The use in these studies of Dic, a potent competitive inhibitor of NAD(P)H [6,39], has supported the proposal that NAD(P)H binding may cause minimal structural changes [46,47] but affects the stability and structural dynamics of the active site, which contributes to the enhancement of catalysis by reducing the reaction free energy barrier(s) and/or promoting quantum tunneling effects [42,45] ( Figure 1B). Interestingly, Dic binding might also allow for the communication of local stability effects between active sites during the catalytic cycle [42,48,49].…”

“…Some reaction steps require the enol or enolate tautomer. (B) Changes in NQO1 structural dynamics upon Dic binding from hydrogen-deuterium exchange (HDX) analysis [42]. The upper panel shows the position of the FAD, Y156 and H162 (involved in the stabilization of FADH2) and Y127 and Y129 (critical for Dic and NAD+ binding).…”

“…(C) leads to decreased dynamics in residues covering the whole active site, particularly regarding the inhibitor and the FAD binding sites that may contribute to optimizing HT from NAD(P)H and FADH2. (D) ∆%Dav is a simple stability metric that refers to the averaged maximal difference in HDX kinetics between two given ligation states according to [42], and a negative value for this parameter reflects an increase in local stability for a given protein segment upon ligand binding (i.e., either HDX is slower and/or its amplitude is reduced upon ligand binding, thus reflecting a locally stabilizing effect upon ligand binding). Note that residue numbering follows the full-length sequence of the protein.…”