Structural basis of the pleiotropic and specific phenotypic consequences of missense mutations in the multifunctional NAD(P)H:quinone oxidoreductase 1 and their pharmacological rescue

Abstract

The multifunctional nature of human flavoproteins is critically linked to their ability to populate multiple conformational states. Ligand binding, post-translational modifications and disease-associated mutations can reshape this functional landscape, although the structure-function relationships of these effects are not well understood. Herein, we characterized the structural and functional consequences of two mutations (the cancer-associated P187S and the phosphomimetic S82D) on different ligation states which are relevant to flavin binding, intracellular stability and catalysis of the disease-associated NQO1 flavoprotein. We found that these mutations affected the stability locally and their effects propagated differently through the protein structure depending both on the nature of the mutation and the ligand bound, showing directional preference from the mutated site and leading to specific phenotypic manifestations in different functional traits (FAD binding, catalysis and inhibition, intracellular stability and pharmacological response to ligands). Our study thus supports that pleitropic effects of disease-causing mutations and phosphorylation events on human flavoproteins may be caused by long-range structural propagation of stability effects to different functional sites that depend on the ligation-state and site-specific perturbations. Our approach can be of general application to investigate these pleiotropic effects at the flavoproteome scale in the absence of high-resolution structural models.

Targeting HIF-1α Function in Cancer through the Chaperone Action of NQO1: Implications of Genetic Diversity of NQO1

“…Thus, we will focus in this section on the structural and dynamic consequences of ligand binding to NQO1 in an attempt to obtain some insight into their regulatory effects on NQO1 interactions with protein partners (always from an NQO1 point of view). NQO1 apo exists as a stable and expanded dimer characterized by significantly large conformational flexibility [ 65 , 67 , 68 , 69 , 111 , 128 , 129 , 130 , 131 ]. Although no high resolution structural model is available for this state, recent kinetic studies using hydrogen–deuterium exchange mass spectrometry (HDXMS) have identified a minimal stable core that holds the protein dimer, while most of the protein exists forming a highly dynamic structural ensemble, including the FAD and substrate binding sites in non-competent states for binding [ 132 ].…”

“…Although no high resolution structural model is available for this state, recent kinetic studies using hydrogen–deuterium exchange mass spectrometry (HDXMS) have identified a minimal stable core that holds the protein dimer, while most of the protein exists forming a highly dynamic structural ensemble, including the FAD and substrate binding sites in non-competent states for binding [ 132 ]. The local stability of this state is very sensitive to mutations [ 131 ]. This remarkable conformational flexibility makes NQO1 apo likely the most relevant state to understand the intracellular stability of NQO1, since the flexible CTD acts as an initiation site for rapid degradation through ubiquitin-dependent and -independent proteasomal pathways [ 88 , 117 , 131 ].…”

“…The local stability of this state is very sensitive to mutations [ 131 ]. This remarkable conformational flexibility makes NQO1 apo likely the most relevant state to understand the intracellular stability of NQO1, since the flexible CTD acts as an initiation site for rapid degradation through ubiquitin-dependent and -independent proteasomal pathways [ 88 , 117 , 131 ]. It is plausible that this unstable and highly dynamic NQO1 apo state is not capable of interacting with NQO1 protein partners, or at least, does so with lower affinity [ 67 ].…”

“…Binding of FAD triggers a large conformational change leading to compaction of the protein dimer, an increase in ordered secondary structure, overall conformational stabilization and a large decrease of protein dynamics that is sensed in almost the entire protein structure [ 67 , 68 , 69 , 78 , 111 , 128 , 129 , 131 , 132 ]. This NQO1 holo state is amenable for high-resolution structural studies [ 70 ] ( Figure 6 A).…”

“…The binding of dicoumarol to wild-type (WT) NQO1 causes strong effects on NQO1 protein dynamics, affecting the local stability of the protein core, and these effects propagate through long distances in the protein structure ( Figure 6 B). Some of these effects are sensed by the CTD ( Figure 6 B), which may explain how dicoumarol binding mildly increases the thermodynamic stability of this domain by about 1.5 kJ·mol −1 [ 69 , 131 ]. Interestingly, a significant fraction of the residues whose stabilities are affected by dicoumarol binding appear on the protein surface and far from the ligand-binding site ( Figure 6 C).…”