Conformational dynamics is key to understanding loss-of-function of NQO1 cancer-associated polymorphisms and its correction by pharmacological ligands

Abstract

Protein dynamics is essential to understand protein function and stability, even though is rarely investigated as the origin of loss-of-function due to genetic variations. Here, we use biochemical, biophysical, cell and computational biology tools to study two loss-of-function and cancer-associated polymorphisms (p.R139W and p.P187S) in human NAD(P)H quinone oxidoreductase 1 (NQO1), a FAD-dependent enzyme which activates cancer pro-drugs and stabilizes several oncosuppressors. We show that p.P187S strongly destabilizes the NQO1 dimer in vitro and increases the flexibility of the C-terminal domain, while a combination of FAD and the inhibitor dicoumarol overcome these alterations. Additionally, changes in global stability due to polymorphisms and ligand binding are linked to the dynamics of the dimer interface, whereas the low activity and affinity for FAD in p.P187S is caused by increased fluctuations at the FAD binding site. Importantly, NQO1 steady-state protein levels in cell cultures correlate primarily with the dynamics of the C-terminal domain, supporting a directional preference in NQO1 proteasomal degradation and the use of ligands binding to this domain to stabilize p.P187S in vivo. In conclusion, protein dynamics are fundamental to understanding loss-of-function in p.P187S, and to develop new pharmacological therapies to rescue this function.The study of NAD(P)H quinone oxidoreductase 1 (NQO1, EC 1.6.5.2) polymorphisms is particularly interesting due to its enhanced expression in several types of cancer 1-3 . NQO1 is a dimeric, two-domain FAD-dependent enzyme (Fig. 1A) which catalyses the two electron reduction of quinones and related substrates through an enzyme-substituted mechanism in which NAD(P)H enters the active site, reduces the FAD and exits as the oxidised form, allowing the subsequent substrate binding and reduction by the FADH 2 4 . Primarily, NQO1 avoids the formation of reactive semiquinones, maintains antioxidants such as α -tocopherol and ubiquinone in their reduced state, and also activates some anticancer bioreductive drugs (e.g. mitomycin C (MMC) and a MMC analogue, EO9) 4,5 . Additionally, NQO1 interacts with tumour suppressors such as p53 and p73 and stabilizes them towards proteasomal degradation 6-9 , while its interaction with the 20S proteasome prevents the degradation of a plethora of proteins with intrinsically disordered regions, including several cell cycle regulators, tumor suppressors and apoptotic proteins 10 .

Site-to-site interdomain communication may mediate different loss-of-function mechanisms in a cancer-associated NQO1 polymorphism

“…The highly dynamic CTD of p.P187S appears to act as an efficient initiation site for its proteasomal degradation24. Consistently, removal of the CTD had significant effects on protein levels and degradation rates upon expression in an eukaryotic expression cell free system (Fig.…”

“…To investigate whether CTD removal could intefere with the dynamic communication between p.P187S and the FAD binding sites, we performed MD simulations on Δ50-NQO1 variants, and analyzed their backbone dynamics using two relevant parameters24: B-factors (Fig. 7) and dihedral entropies (Figure S5).…”

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

“…This polymorphism shows a high frequency in human population and is significantly associated with increased cancer risk . The P187S polymorphism is known to abolish NQO1 activity by strongly decreasing FAD‐binding affinity and accelerating protein turnover by the proteasome . Intriguingly, the X‐ray crystallographic structure of P187S in the FAD‐bound (holo) state only showed subtle local rearrangements , which cannot explain the long‐range communication of its effect to the N‐terminal domain (NTD, where the FAD‐binding site is placed) or the C‐terminal domain (CTD) .…”

Experimental and computational evidence on conformational fluctuations as a source of catalytic defects in genetic diseases

“…S2) and to ensure that the proteolysis step X →F is ratelimiting, and thus, the overall proteolysis rate constant k p ≈K·k 8,41,48 . Proteolysis of GALE under native conditions is well-described by the following mechanism: N ⇔X⇒F, where X is the cleavable state in equilibrium with the native state (N), and undergoes proteolytic cleavage to produce a proteolyzed state F. The step N ↔X is governed by a equilibrium constant K, while the step X →F is determined by an intrinsic proteolysis rate constant k. To provide insight into the conformation of X, we have measured proteolysis kinetics in the presence of low urea concentrations.…”