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

Abstract

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.

Inhibitors of HSP90 in melanoma

“…Cell sensitivity to ansamycin inhibitors of HSP90 was also associated with expression of NQO1 encoding NAD(P)H:quinone oxidoreductase 1 that converted these compounds to hydroquinone to enhance their activity by increasing hydrogen bonding [99]. It was reported that NQO1 P187S variant exerted diminished activity compared with wild-type NQO1 [99], but genetic alterations affecting a His 80 residue in this protein could compensate for P187S substitution [100]. Loss of NQO1 expression and acquisition of NQO1 P187S variant contributed to the development of resistance to 17-AAG [101].…”

Targeting HIF-1α Function in Cancer through the Chaperone Action of NQO1

“…NQO1apo exists as a stable and expanded dimer characterized by significantly large conformational flexibility [76,78,79,80,119,130,131,132]. 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 [129].…”

“…Binding of FAD triggers a large conformational change leading to compaction of the protein dimer, increase in ordered secondary structure, overall conformational stabilization and a large decrease of protein dynamics that is sensed in almost the entire protein structure [78,79,80,119,129,130,131,133]. This NQO1holo state is amenable for high-resolution structural studies [81]( Figure 6A).…”

“…Binding of dicoumarol (or NAD + ) causes small structural rearrangements in the protein structure [75,78,81,131,134] (Figure 6A). Therefore, from NQO1 structural point of view, it is not straightforward to understand how NQO1dic prevents binding to or largely decrease binding affinity for macromolecular partners with subsequent effects on the stability of these partners [78,127].…”

“…This seems to be paradoxical, because P187 is fully buried in the protein structure, close to the NQO1 dimer interface and belongs to the minimally stable core of NQO1apo, and thus, a mutation to serine should have catastrophic effects on protein structure and function [129,136]. Detailed biochemical, biophysical, computational and mutational studies have shown that the effects of p.P187S are pleiotropic in the structure, function and stability of NQO1apo and NQO1holo, highlighting the critical role of propagation of the local stability effects due to p.P187S to long distances in the protein structure and affecting the dynamics and stability of critical regions of NQO1 function [78,79,90,119,120,122,131,133,136,137]. These studies on p.P187S have also supported that different functional sites located far (over 10-20 Å ) in the structure are functionally and energetically coupled, and thus, we might modify the interaction of NQO1 with its partners by using an allosteric site (i.e.…”