Thermodynamics of cooperative binding of FAD to human NQO1: Implications to understanding cofactor-dependent function and stability of the flavoproteome

Clavería-Gimeno, Rafael; Velázquez‐Campoy, Adrián; Pey, Ángel L.

doi:10.1016/j.abb.2017.10.020

Cited by 27 publications

(59 citation statements)

References 35 publications

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“…It might be important in the mechanism of protein chaperoning, as noted above. Another possibility is that the negative cooperativity in FAD binding coincidentally affects interaction with inhibitors such as dicoumarol . If this were the case, it seems likely that negative cooperativity is important in the protein's redox‐sensing role, potentially enabling it to act over a wide range of cellular FAD concentrations.…”

Section: Resultsmentioning

confidence: 99%

“…Although the C‐terminal domain (CTD) is not directly involved in forming the active site, removal of this part of the protein dramatically reduces the affinity for FAD and, consequently, catalytic activity . The binding of FAD to one active site reduces the affinity at the other . This negative cooperativity of cofactor binding might have implications for catalysis and the binding of other ligands .…”

Section: Introductionmentioning

confidence: 99%

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Negative Cooperativity in NAD(P)H Quinone Oxidoreductase 1 (NQO1)

et al. 2019

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NAD(P)H quinone oxidoreductase‐1 (NQO1) is a homodimeric protein that acts as a detoxifying enzyme or as a chaperone protein. Dicourmarol interacts with NQO1 at the NAD(P)H binding site and can both inhibit enzyme activity and modulate the interaction of NQO1 with other proteins. We show that the binding of dicoumarol and related compounds to NQO1 generates negative cooperativity between the monomers. This does not occur in the presence of the reducing cofactor, NAD(P)H, alone. Alteration of Gly150 (but not Gly149 or Gly174) abolished the dicoumarol‐induced negative cooperativity. Analysis of the dynamics of NQO1 with the Gaussian network model indicates a high degree of collective motion by monomers and domains within NQO1. Ligand binding is predicted to alter NQO1 dynamics both proximal to the ligand binding site and remotely, close to the second binding site. Thus, drug‐induced modulation of protein motion might contribute to the biological effects of putative inhibitors of NQO1.

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Section: Resultsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Negative Cooperativity in NAD(P)H Quinone Oxidoreductase 1 (NQO1)

et al. 2019

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“…Fluorescence titrations were carried out at 25 • C using 1 × 0.3 cm path-length cuvettes in a Cary Eclipse spectrofluorimeter (Agilent Technologies, Madrid, Spain). Experiments were carried out in 20 mM K-phosphate, pH 7.4, to decrease the binding affinity of NQO1 for FAD and to allow more accurate characterization of the affinity in tightly binding variants [42]. Then, 20 µL of a 12.5 µM NQO1 stock solution (in subunit) was mixed with 0-500 µL of FAD 10 µM and the corresponding volume of buffer was added to yield a 1 mL final volume.…”

Section: In Vitro Characterization Of Purified Proteinsmentioning

confidence: 99%

“…We have recently used the enzyme NAD(P)H quinone oxidoreductase 1 (NQO1; EC 1.6.5.2) to investigate how missense mutations may affect different functional features in a multifunctional human protein [19,[39][40][41][42][43][44][45]. NQO1 folds into functional dimers of 62 kDa [46,47] and each monomer has 274 residues divided into two domains: an N-terminal domain (NTD, residues 1-225) that contains a tightly bound FAD and most of the catalytic site and the monomer:monomer interface (MMI), and a C-terminal domain (CTD, residues 225-274) that completes the MMI and the active site [44,46,[48][49][50][51].…”

Section: Introductionmentioning

confidence: 99%

Naturally-Occurring Rare Mutations Cause Mild to Catastrophic Effects in the Multifunctional and Cancer-Associated NQO1 Protein

Pacheco-García

Cano-Muñoz

Sánchez-Ramos

et al. 2020

JPM

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The functional and pathological implications of the enormous genetic diversity of the human genome are mostly unknown, primarily due to our unability to predict pathogenicity in a high-throughput manner. In this work, we characterized the phenotypic consequences of eight naturally-occurring missense variants on the multifunctional and disease-associated NQO1 protein using biophysical and structural analyses on several protein traits. Mutations found in both exome-sequencing initiatives and in cancer cell lines cause mild to catastrophic effects on NQO1 stability and function. Importantly, some mutations perturb functional features located structurally far from the mutated site. These effects are well rationalized by considering the nature of the mutation, its location in protein structure and the local stability of its environment. Using a set of 22 experimentally characterized mutations in NQO1, we generated experimental scores for pathogenicity that correlate reasonably well with bioinformatic scores derived from a set of commonly used algorithms, although the latter fail to semiquantitatively predict the phenotypic alterations caused by a significant fraction of mutations individually. These results provide insight into the propagation of mutational effects on multifunctional proteins, the implementation of in silico approaches for establishing genotype-phenotype correlations and the molecular determinants underlying loss-of-function in genetic diseases.

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“…The intracellular effects of p.P187S can be ascribed to changes in its stability and activity, respectively. This variant decreases by 10-to 40-fold the affinity for FAD thus promoting the population of NQO1apo [79,120,122,133,136,142]. Unlike WT NQO1, p.P187S in both NQO1holo and NQO1apo are degraded similarly, due to a strong thermodynamic destabilization of the CTD in NQO1holo that triggers ubiquitination and degradation [78,79,116,119,120,122].…”

Section: Mutations and Polymorphisms In Nqo1 Disease And Protein Intmentioning

confidence: 99%

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

Salido¹,

Timson²,

Betancor-Fernández³

et al. 2020

Preprint

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HIF-1α is a master regulator of oxygen homeostasis involved in different stages of cancer development. Thus, HIF-1α inhibition represents an interesting target for anti-cancer therapy. It was recently shown that HIF-1α interaction with NQO1 inhibits its proteasomal degradation, thus suggesting that targeting the stability of NQO1 could led to destabilization of HIF-1α as a therapeutic approach. Since the molecular interactions of NQO1 with HIF-1α are beginning to be unraveled, we review here our current knowledge on the intracellular functions and stability of NQO1, its pharmacological modulation by small ligands, and the molecular determinants of its roles as a chaperone of many different proteins including cancer-associated factors such as p53 and p73α. This knowledge is then discussed in the context of potentially targeting the intracellular stability of HIF-1α by acting on its chaperone, NQO1. This could result in novel anti-cancer therapies.

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Thermodynamics of cooperative binding of FAD to human NQO1: Implications to understanding cofactor-dependent function and stability of the flavoproteome

Cited by 27 publications

References 35 publications

Negative Cooperativity in NAD(P)H Quinone Oxidoreductase 1 (NQO1)

Negative Cooperativity in NAD(P)H Quinone Oxidoreductase 1 (NQO1)

Naturally-Occurring Rare Mutations Cause Mild to Catastrophic Effects in the Multifunctional and Cancer-Associated NQO1 Protein

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

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Thermodynamics of cooperative binding of FAD to human NQO1: Implications to understanding cofactor-dependent function and stability of the flavoproteome

Cited by 27 publications

References 35 publications

Negative Cooperativity in NAD(P)H Quinone Oxidoreductase 1 (NQO1)

Negative Cooperativity in NAD(P)H Quinone Oxidoreductase 1 (NQO1)

Naturally-Occurring Rare Mutations Cause Mild to Catastrophic Effects in the Multifunctional and Cancer-Associated NQO1 Protein

Targeting HIF-1&alpha; Function in Cancer through the Chaperone Action of NQO1

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