Catalytic competence, structure and stability of the cancer‐associated R139W variant of the human NAD(P)H:quinone oxidoreductase 1 (NQO1)

Abstract

The human NAD(P)H:quinone oxidoreductase 1 (NQO1; EC 1.6.99.2) is an essential enzyme in the antioxidant defence system. Furthermore, NQO1 protects tumour suppressors like p53, p33ING1b and p73 from proteasomal degradation. The activity of NQO1 is also exploited in chemotherapy for the activation of quinone-based treatments. Various single nucleotide polymorphisms are known, such as NQO1*2 and NQO1*3 yielding protein variants of NQO1 with single amino acid replacements, i.e. P187S and R139W, respectively. While the former NOQ1 variant is linked to a higher risk for specific kinds of cancer, the role, if any, of the arginine 139 to tryptophan exchange in disease development remains obscure. On the other hand, mitomycin C-resistant human colon cancer cells were shown to harbour the NQO1*3 variant resulting in substantially reduced enzymatic activity. However, the molecular cause for this decrease remains unclear. In order to resolve this issue, recombinant NQO1 R139W has been characterized biochemically and structurally. In this report, we show by X-ray crystallography and 2D-NMR spectroscopy that this variant adopts the same structure both in the crystal as well as in solution. Furthermore, the kinetic parameters obtained for the variant are similar to those reported for the wild-type protein. Similarly, thermostability of the variant was only slightly affected by the amino acid replacement. Therefore, we conclude that the previously reported effects in human cancer cells cannot be attributed to protein stability or enzyme activity. Instead, it appears that loss of exon 4 during maturation of a large fraction of pre-mRNA is the major reason of the observed lack of enzyme activity and hence reduced activation of quinone-based chemotherapeutics.

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

“…5A,D,G). In agreement with a recent study using WT NQO1 [18], FAD binding causes a significant compaction of the solution ensemble, as shown by the decrease in R g and D max values in all three variants ( Fig. 5B,E,H).…”

“…Therefore, a combination of structural and dynamic analysis using a wide variety of techniques (titration calorimetry, proteolysis, MD simulations, SAXS, FTIR and CD [17] and this work) support the existence of significant conformational plasticity in the apo-state of NQO1 in response to single amino acid exchanges. This integrated analysis completes and refines previous information obtained from high-resolution structural techniques such as X-ray crystallography or NMR spectroscopy [3,17,18]. We also provide here a detailed characterization of the suppressor effect of the consensus amino acid Arg80, which has recently diverged to His80 along primate evolution (about 30 million years ago), potentially predisposing human NQO1 towards cancer-associated inactivation [17].…”

“…A ). Therefore, the apo‐state is mostly populated by noncompetent binding states constituting a very flexible and dynamic ensemble (as seen by the large sensitivity of apo‐proteins towards partial proteolysis, and also supported by NMR, MD and SAXS analyses and this work). In contrast, the binding competent states may structurally resemble the protein in the bound conformation (i.e.…”

“…Importantly, experimental Δ C p values are larger than the values calculated from crystal structures of the FAD‐bound, holo‐state (Fig. E), confirming a departure from a lock‐and‐key binding mechanism and a remarkable conformational change upon FAD binding . In this context, the different Δ H at 25 °C and Δ C p for FAD binding between H80R/P187S and P187S (Fig.…”

A small molecule chaperone rescues the stability and activity of a cancer‐associated variant of NAD(P)H:quinone oxidoreductase 1 in vitro

“…However, the P187S variant featured an increase in both radius of gyration (R g ) and maximum distance (D max ) compared to the wild-type protein (R g of 2.60 and 3.04 nm, D max of 8 and 11 nm for wildtype and the variant protein, respectively). This indicates a more extended conformation of the P187S variant dimer compared to the wild-type protein, as similar observations have previously been reported [13,36,37]. The addition of BPPSA led to a decrease in both R g and D max for the P187S variant, whereas no significant changes were observed for the wild-type protein (R g of 3.04 and 2.69 nm and D max of 11 and 9 nm, for the P187S variant in the absence and presence of BPPSA, respectively).…”