Collapse of the native structure caused by a single amino acid exchange in human NAD(P)H:quinone oxidoreductase¹

Abstract

Human NAD(P)H:quinone oxidoreductase 1 (NQO1) is essential for the antioxidant defense system, stabilization of tumor suppressors (e.g. p53, p33, and p73), and activation of quinone-based chemotherapeutics. Overexpression of NQO1 in many solid tumors, coupled with its ability to convert quinone-based chemotherapeutics into potent cytotoxic compounds, have made it a very attractive target for anticancer drugs. A naturally occurring single-nucleotide polymorphism (C609T) leading to an amino acid exchange (P187S) has been implicated in the development of various cancers and poor survival rates following anthracyclin-based adjuvant chemotherapy. Despite its importance for cancer prediction and therapy, the exact molecular basis for the loss of function in NQO1 P187S is currently unknown. Therefore, we solved the crystal structure of NQO1 P187S. Surprisingly, this structure is almost identical to NQO1. Employing a combination of NMR spectroscopy and limited proteolysis experiments, we demonstrated that the single amino acid exchange destabilized interactions between the core and C-terminus, leading to depopulation of the native structure in solution. This collapse of the native structure diminished cofactor affinity and led to a less competent FAD-binding pocket, thus severely compromising the catalytic capacity of the variant protein. Hence, our findings provide a rationale for the loss of function in NQO1 P187S with a frequently occurring single-nucleotide polymorphism.

Recognition of enzymes lacking bound cofactor by protein quality control

“…We wondered whether the FAD-free wild-type NQO1 and the mutant with reduced affinity for the cofactor are recognized through the same structural determinants. Recent analysis of the mutant NQO1 indicated that the C-terminal tail loses its proper association with the core domain, especially when the NADH-binding pocket is not occupied (22,29). A protease sensitivity assay combined with Western blotting using a C-terminal tail-reacting antibody confirmed the rapid loss of the C-terminus by the mutant and the wild-type apoNQO1 (Fig.…”

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

“…However, molecular dynamics (MD) simulations, biophysical experiments and expression analyses have supported the hypothesis that p.P187S inactivates and destabilizes NQO1 through dynamic changes in the apo-state, in particular on two functionally and structurally distant sites (Fig. 1D): the FAD binding site, associated with enzyme inactivation; and the CTD, associated with enhanced proteasomal degradation of p.P187S222431.…”

“…NQO1 also interacts with and stabilizes cancer-associated transcription factors such as p53 and p73α27282930, even though the structural basis of this intracellular interaction is unknown. Although p.P187S strongly reduces NQO1 levels and activity in vivo , crystallographic analyses of holo-p.P187S have shown no significant effect on functional or structural sites such as FAD binding site, the CTD or the monomer:monomer interface31. However, molecular dynamics (MD) simulations, biophysical experiments and expression analyses have supported the hypothesis that p.P187S inactivates and destabilizes NQO1 through dynamic changes in the apo-state, in particular on two functionally and structurally distant sites (Fig.…”

Dicoumarol: A Drug which Hits at Least Two Very Different Targets in Vitamin K Metabolism

“…A rarer allele codes for p.R139W (NQO1*3; rs1131341) which is associated with childhood acute lymphoblastic leukaemia [100]. The fundamental biochemical cause of the loss of NQO1 activity in both variants is decreased stability of the protein and, in the case of p.P187S, consequent loss of the FAD cofactor [98,[101][102][103].…”