Structural dynamics and functional cooperativity of human NQO1 by ambient temperature serial crystallography and simulations.

The human NQO1 (hNQO1) is a flavin adenine nucleotide (FAD)-dependent oxidoreductase that catalyzes the two-electron reduction of quinones to hydroquinones, being essential for the antioxidant defense system, stabilization of tumor suppressors, and activation of quinone-based chemotherapeutics. Moreover, it is overexpressed in several tumors, which makes it an attractive cancer drug target. To decipher new structural insights into the flavin reductive half-reaction of the catalytic mechanism of hNQO1, we have carried serial crystallography experiments at new ID29 beamline of the ESRF to determine, to the best of our knowledge, the first structure of the hNQO1 in complex with NADH. We have also performed molecular dynamics simulations of free hNQO1 and in complex with NADH. This is the first structural evidence that the hNQO1 functional cooperativity is driven by structural communication between the active sites through long-range propagation of cooperative effects across the hNQO1 structure. Both structural results and MD simulations have supported that the binding of NADH significantly decreases protein dynamics and stabilizes hNQO1 especially at the dimer core and interface. Altogether, these results pave the way for future time-resolved studies, both at x-ray free-electron lasers and synchrotrons, of the dynamics of hNQO1 upon binding to NADH as well as during the FAD cofactor reductive half-reaction. This knowledge will allow us to reveal unprecedented structural information of the relevance of the dynamics during the catalytic function of hNQO1.

Insights into the pathogenesis of primary hyperoxaluria type I from the structural dynamics of alanine:glyoxylate aminotransferase variants.

Primary hyperoxaluria type I (PH1) is caused by deficient alanine:glyoxylate aminotransferase (AGT) activity. PH1-causing mutations in AGT lead to protein mistargeting and aggregation. Here, we use hydrogen-deuterium exchange (HDX) to characterize the wild-type (WT), the LM (a polymorphism frequent in PH1 patients) and the LM G170R (the most common mutation in PH1) variants of AGT. We provide the first experimental analysis of AGT structural dynamics, showing that stability is heterogeneous in the native state and providing a blueprint for frustrated regions with potentially functional relevance. The LM and LM G170R variants only show local destabilization. Enzymatic transamination of the pyridoxal 5-phosphate cofactor bound to AGT hardly affects stability. Our study, thus, supports that AGT misfolding is not caused by dramatic effects on structural dynamics.

Structural dynamics at the active site of the cancer-associated flavoenzyme NQO1 probed by chemical modification with PMSF.

A large conformational heterogeneity of human NAD(P)H:quinone oxidoreductase 1 (NQO1), a flavoprotein associated with various human diseases, has been observed to occur in the catalytic site of the enzyme. Here, we report the X-ray structure of NQO1 with phenylmethylsulfonyl fluoride (PMSF) at 1.6 Å resolution. Activity assays confirmed that, despite being covalently bound to the Tyr128 residue at the catalytic site, PMSF did not abolish NQO1 activity. This may indicate that the PMSF molecule does not reduce the high flexibility of Tyr128, thus allowing NADH and DCPIP substrates to bind to the enzyme. Our results show that targeting Tyr128, a key residue in NQO1 function, with small covalently bound molecules could possibly not be a good drug discovery strategy to inhibit this enzyme.

Modular droplet injector for sample conservation providing new structural insight for the conformational heterogeneity in the disease-associated NQO1 enzyme.

Droplet injection strategies are a promising tool to reduce the large amount of sample consumed in serial femtosecond crystallography (SFX) measurements at X-ray free electron lasers (XFELs) with continuous injection approaches. Here, we demonstrate a new modular microfluidic droplet injector (MDI) design that was successfully applied to deliver microcrystals of the human NAD(P)H:quinone oxidoreductase 1 (NQO1) and phycocyanin. We investigated droplet generation conditions through electrical stimulation for both protein samples and implemented hardware and software components for optimized crystal injection at the Macromolecular Femtosecond Crystallography (MFX) instrument at the Stanford Linac Coherent Light Source (LCLS). Under optimized droplet injection conditions, we demonstrate that up to 4-fold sample consumption savings can be achieved with the droplet injector. In addition, we collected a full data set with droplet injection for NQO1 protein crystals with a resolution up to 2.7 Å, leading to the first room-temperature structure of NQO1 at an XFEL. NQO1 is a flavoenzyme associated with cancer, Alzheimer's and Parkinson's disease, making it an attractive target for drug discovery. Our results reveal for the first time that residues Tyr128 and Phe232, which play key roles in the function of the protein, show an unexpected conformational heterogeneity at room temperature within the crystals. These results suggest that different substates exist in the conformational ensemble of NQO1 with functional and mechanistic implications for the enzyme's negative cooperativity through a conformational selection mechanism. Our study thus demonstrates that microfluidic droplet injection constitutes a robust sample-conserving injection method for SFX studies on protein crystals that are difficult to obtain in amounts necessary for continuous injection, including the large sample quantities required for time-resolved mix-and-inject studies.

Phenotypic Modulation of Cancer-Associated Antioxidant NQO1 Activity by Post-Translational Modifications and the Natural Diversity of the Human Genome.

Human NAD(P)H:quinone oxidoreductase 1 (hNQO1) is a multifunctional and antioxidant stress protein whose expression is controlled by the Nrf2 signaling pathway. hNQO1 dysregulation is associated with cancer and neurological disorders. Recent works have shown that its activity is also modulated by different post-translational modifications (PTMs), such as phosphorylation, acetylation and ubiquitination, and these may synergize with naturally-occurring and inactivating polymorphisms and mutations. Herein, I describe recent advances in the study of the effect of PTMs and genetic variations on the structure and function of hNQO1 and their relationship with disease development in different genetic backgrounds, as well as the physiological roles of these modifications. I pay particular attention to the long-range allosteric effects exerted by PTMs and natural variation on the multiple functions of hNQO1.

Molecular Mechanisms, Genotype-Phenotype Correlations and Patient-Specific Treatments in Inherited Metabolic Diseases.

Advances in DNA sequencing technologies are revealing a vast genetic heterogeneity in human population, which may predispose to metabolic alterations if the activity of metabolic enzymes is affected [...].

Counterintuitive structural and functional effects due to naturally occurring mutations targeting the active site of the disease-associated NQO1 enzyme.

Our knowledge on the genetic diversity of the human genome is exponentially growing. However, our capacity to establish genotype-phenotype correlations on a large scale requires a combination of detailed experimental and computational work. This is a remarkable task in human proteins which are typically multifunctional and structurally complex. In addition, mutations often prevent the determination of mutant high-resolution structures by X-ray crystallography. We have characterized here the effects of five mutations in the active site of the disease-associated NQO1 protein, which are found either in cancer cell lines or in massive exome sequencing analysis in human population. Using a combination of H/D exchange, rapid-flow enzyme kinetics, binding energetics and conformational stability, we show that mutations in both sets may cause counterintuitive functional effects that are explained well by their effects on local stability regarding different functional features. Importantly, mutations predicted to be highly deleterious (even those affecting the same protein residue) may cause mild to catastrophic effects on protein function. These functional effects are not well explained by current predictive bioinformatic tools and evolutionary models that account for site conservation and physicochemical changes upon mutation. Our study also reinforces the notion that naturally occurring mutations not identified as disease-associated can be highly deleterious. Our approach, combining protein biophysics and structural biology tools, is readily accessible to broadly increase our understanding of genotype-phenotype correlations and to improve predictive computational tools aimed at distinguishing disease-prone against neutral missense variants in the human genome.

Phosphorylation of Thr9 Affects the Folding Landscape of the N-Terminal Segment of Human AGT Enhancing Protein Aggregation of Disease-Causing Mutants.

The mutations G170R and I244T are the most common disease cause in primary hyperoxaluria type I (PH1). These mutations cause the misfolding of the AGT protein in the minor allele AGT-LM that contains the P11L polymorphism, which may affect the folding of the N-terminal segment (NTT-AGT). The NTT-AGT is phosphorylated at T9, although the role of this event in PH1 is unknown. In this work, phosphorylation of T9 was mimicked by introducing the T9E mutation in the NTT-AGT peptide and the full-length protein. The NTT-AGT conformational landscape was studied by circular dichroism, NMR, and statistical mechanical methods. Functional and stability effects on the full-length AGT protein were characterized by spectroscopic methods. The T9E and P11L mutations together reshaped the conformational landscape of the isolated NTT-AGT peptide by stabilizing ordered conformations. In the context of the full-length AGT protein, the T9E mutation had no effect on the overall AGT function or conformation, but enhanced aggregation of the minor allele (LM) protein and synergized with the mutations G170R and I244T. Our findings indicate that phosphorylation of T9 may affect the conformation of the NTT-AGT and synergize with PH1-causing mutations to promote aggregation in a genotype-specific manner. Phosphorylation should be considered a novel regulatory mechanism in PH1 pathogenesis.

Loss of stability and unfolding cooperativity in hPGK1 upon gradual structural perturbation of its N-terminal domain hydrophobic core.

Phosphoglycerate kinase has been a model for the stability, folding cooperativity and catalysis of a two-domain protein. The human isoform 1 (hPGK1) is associated with cancer development and rare genetic diseases that affect several of its features. To investigate how mutations affect hPGK1 folding landscape and interaction networks, we have introduced mutations at a buried site in the N-terminal domain (F25 mutants) that either created cavities (F25L, F25V, F25A), enhanced conformational entropy (F25G) or introduced structural strain (F25W) and evaluated their effects using biophysical experimental and theoretical methods. All F25 mutants folded well, but showed reduced unfolding cooperativity, kinetic stability and altered activation energetics according to the results from thermal and chemical denaturation analyses. These alterations correlated well with the structural perturbation caused by mutations in the N-terminal domain and the destabilization caused in the interdomain interface as revealed by H/D exchange under native conditions. Importantly, experimental and theoretical analyses showed that these effects are significant even when the perturbation is mild and local. Our approach will be useful to establish the molecular basis of hPGK1 genotype-phenotype correlations due to phosphorylation events and single amino acid substitutions associated with disease.

Different phenotypic outcome due to site-specific phosphorylation in the cancer-associated NQO1 enzyme studied by phosphomimetic mutations.

Protein phosphorylation is a common phenomenon in human flavoproteins although the functional consequences of this site-specific modification are largely unknown. Here, we evaluated the effects of site-specific phosphorylation (using phosphomimetic mutations at sites S40, S82 and T128) on multiple functional aspects as well as in the structural stability of the antioxidant and disease-associated human flavoprotein NQO1 using biophysical and biochemical methods. In vitro biophysical studies revealed effects of phosphorylation at different sites such as decreased binding affinity for FAD and structural stability of its binding site (S82), conformational stability (S40 and S82) and reduced catalytic efficiency and functional cooperativity (T128). Local stability measurements by H/D exchange in different ligation states provided structural insight into these effects. Transfection of eukaryotic cells showed that phosphorylation at sites S40 and S82 may reduce steady-levels of NQO1 protein by enhanced proteasome-induced degradation. We show that site-specific phosphorylation of human NQO1 may cause pleiotropic and counterintuitive effects on this multifunctional protein with potential implications for its relationships with human disease. Our approach allows to establish relationships between site-specific phosphorylation, functional and structural stability effects in vitro and inside cells paving the way for more detailed analyses of phosphorylation at the flavoproteome scale.

Allosteric Communication in the Multifunctional and Redox NQO1 Protein Studied by Cavity-Making Mutations.

Allosterism is a common phenomenon in protein biochemistry that allows rapid regulation of protein stability; dynamics and function. However, the mechanisms by which allosterism occurs (by mutations or post-translational modifications (PTMs)) may be complex, particularly due to long-range propagation of the perturbation across protein structures. In this work, we have investigated allosteric communication in the multifunctional, cancer-related and antioxidant protein NQO1 by mutating several fully buried leucine residues (L7, L10 and L30) to smaller residues (V, A and G) at sites in the N-terminal domain. In almost all cases, mutated residues were not close to the FAD or the active site. Mutations L→G strongly compromised conformational stability and solubility, and L30A and L30V also notably decreased solubility. The mutation L10A, closer to the FAD binding site, severely decreased FAD binding affinity (≈20 fold vs. WT) through long-range and context-dependent effects. Using a combination of experimental and computational analyses, we show that most of the effects are found in the apo state of the protein, in contrast to other common polymorphisms and PTMs previously characterized in NQO1. The integrated study presented here is a first step towards a detailed structural-functional mapping of the mutational landscape of NQO1, a multifunctional and redox signaling protein of high biomedical relevance.

New salicylic acid derivatives, double inhibitors of glycolate oxidase and lactate dehydrogenase, as effective agents decreasing oxalate production.

The synthesis and biological evaluation of double glycolate oxidase/lactate dehydrogenase inhibitors containing a salicylic acid moiety is described. The target compounds are obtained in an easily scalable two-step synthetic procedure. These compounds showed low micromolar IC50 values against the two key enzymes in the metabolism of glyoxylate. Mechanistically they behave as noncompetitive inhibitors against both enzymes and this fact is supported by docking studies. The biological evaluation also includes in vitro and in vivo assays in hyperoxaluric mice. The compounds are active against the three types of primary hyperoxalurias. Also, possible causes of adverse effects, such as cyclooxygenase inhibition or renal toxicity, have been studied and discarded. Altogether, this makes this chemotype with drug-like structure a good candidate for the treatment of primary hyperoxalurias.

Targeting HIF-1α Function in Cancer through the Chaperone Action of NQO1: Implications of Genetic Diversity of NQO1.

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 the HIF-1α interaction with NQO1 inhibits proteasomal degradation of the former, thus suggesting that targeting the stability and/or function of NQO1 could lead to the destabilization of HIF-1α as a therapeutic approach. Since the molecular interactions of NQO1 with HIF-1α are beginning to be unraveled, in this review we discuss: (1) Structure-function relationships of HIF-1α; (2) our current knowledge on the intracellular functions and stability of NQO1; (3) the pharmacological modulation of NQO1 by small ligands regarding function and stability; (4) the potential effects of genetic variability of NQO1 in HIF-1α levels and function; (5) the molecular determinants of NQO1 as a chaperone of many different proteins including cancer-associated factors such as HIF-1α, p53 and p73α. This knowledge is then further 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, always considering that the substantial genetic variability in NQO1 would likely result in different phenotypic responses among individuals.

A single evolutionarily divergent mutation determines the different FAD-binding affinities of human and rat NQO1 due to site-specific phosphorylation.

The phosphomimetic mutation S82D in the cancer-associated, FAD-dependent human NADP(H):quinone oxidoreductase 1 (hNQO1) causes a decrease in flavin-adenine dinucleotide-binding affinity and intracellular stability. We test in this work whether the evolutionarily recent neutral mutation R80H in the vicinity of S82 may alter the strong functional effects of S82 phosphorylation through electrostatic interactions. We show using biophysical and bioinformatic analyses that the reverse mutation H80R prevents the effects of S82D phosphorylation on hNQO1 by modulating the local stability. Consistently, in rat NQO1 (rNQO1) which contains R80, the effects of phosphorylation were milder, resembling the behaviour found in hNQO1 when this residue was humanized in rNQO1 (by the R80H mutation). Thus, apparently neutral and evolutionarily divergent mutations may determine the functional response of mammalian orthologues towards phosphorylation.

Structural basis of the pleiotropic and specific phenotypic consequences of missense mutations in the multifunctional NAD(P)H:quinone oxidoreductase 1 and their pharmacological rescue.

The multifunctional nature of human flavoproteins is critically linked to their ability to populate multiple conformational states. Ligand binding, post-translational modifications and disease-associated mutations can reshape this functional landscape, although the structure-function relationships of these effects are not well understood. Herein, we characterized the structural and functional consequences of two mutations (the cancer-associated P187S and the phosphomimetic S82D) on different ligation states which are relevant to flavin binding, intracellular stability and catalysis of the disease-associated NQO1 flavoprotein. We found that these mutations affected the stability locally and their effects propagated differently through the protein structure depending both on the nature of the mutation and the ligand bound, showing directional preference from the mutated site and leading to specific phenotypic manifestations in different functional traits (FAD binding, catalysis and inhibition, intracellular stability and pharmacological response to ligands). Our study thus supports that pleitropic effects of disease-causing mutations and phosphorylation events on human flavoproteins may be caused by long-range structural propagation of stability effects to different functional sites that depend on the ligation-state and site-specific perturbations. Our approach can be of general application to investigate these pleiotropic effects at the flavoproteome scale in the absence of high-resolution structural models.

Dimerization Drives Proper Folding of Human Alanine:Glyoxylate Aminotransferase But Is Dispensable for Peroxisomal Targeting.

Peroxisomal matrix proteins are transported into peroxisomes in a fully-folded state, but whether multimeric proteins are imported as monomers or oligomers is still disputed. Here, we used alanine:glyoxylate aminotransferase (AGT), a homodimeric pyridoxal 5'-phosphate (PLP)-dependent enzyme, whose deficit causes primary hyperoxaluria type I (PH1), as a model protein and compared the intracellular behavior and peroxisomal import of native dimeric and artificial monomeric forms. Monomerization strongly reduces AGT intracellular stability and increases its aggregation/degradation propensity. In addition, monomers are partly retained in the cytosol. To assess possible differences in import kinetics, we engineered AGT to allow binding of a membrane-permeable dye and followed its intracellular trafficking without interfering with its biochemical properties. By fluorescence recovery after photobleaching, we measured the import rate in live cells. Dimeric and monomeric AGT displayed a similar import rate, suggesting that the oligomeric state per se does not influence import kinetics. However, when dimerization is compromised, monomers are prone to misfolding events that can prevent peroxisomal import, a finding crucial to predicting the consequences of PH1-causing mutations that destabilize the dimer. Treatment with pyridoxine of cells expressing monomeric AGT promotes dimerization and folding, thus, demonstrating the chaperone role of PLP. Our data support a model in which dimerization represents a potential key checkpoint in the cytosol at the crossroad between misfolding and correct targeting, a possible general mechanism for other oligomeric peroxisomal proteins.

Galactosemia: Towards Pharmacological Chaperones.

Galactosemia is a rare inherited metabolic disease resulting from mutations in the four genes which encode enzymes involved in the metabolism of galactose. The current therapy, the removal of galactose from the diet, is inadequate. Consequently, many patients suffer lifelong physical and cognitive disability. The phenotype varies from almost asymptomatic to life-threatening disability. The fundamental biochemical cause of the disease is a decrease in enzymatic activity due to failure of the affected protein to fold and/or function correctly. Many novel therapies have been proposed for the treatment of galactosemia. Often, these are designed to treat the symptoms and not the fundamental cause. Pharmacological chaperones (PC) (small molecules which correct the folding of misfolded proteins) represent an exciting potential therapy for galactosemia. In theory, they would restore enzyme function, thus preventing downstream pathological consequences. In practice, no PCs have been identified for potential application in galactosemia. Here, we review the biochemical basis of the disease, identify opportunities for the application of PCs and describe how these might be discovered. We will conclude by considering some of the clinical issues which will affect the future use of PCs in the treatment of galactosemia.

Intrinsically disordered protein NUPR1 binds to the armadillo-repeat domain of Plakophilin 1.

Plakophilin 1 (PKP1), a member of the armadillo repeat family of proteins, is a scaffold component of desmosomes, which are key structural components for cell-cell adhesion. However, PKP1 can be also found in the nucleus of several cells. NUPR1 is an intrinsically disordered protein (IDP) that localizes throughout the whole cell, and intervenes in the development and progression of several cancers. In this work, we studied the binding between PKP1 and NUPR1 by using several in vitro biophysical techniques and in cellulo approaches. The interaction occurred with an affinity in the low micromolar range (~10 µM), and involved the participation of at least one of the tryptophan residues of PKP1 (as shown by fluorescence and molecular docking). The binding region of NUPR1, mapped by NMR and molecular modelling, was a polypeptide patch at the 30s region of its sequence. The association between PKP1 and NUPR1 also occurred in cellulo and was localized in the nucleus, as tested by protein ligation assays (PLAs). We hypothesize that NUPR1 plays an active role in carcinogenesis modulating the function of PKP1.

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

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.

Towards Accurate Genotype-Phenotype Correlations in the CYP2D6 Gene.

Establishing accurate and large-scale genotype-phenotype correlations and predictions of individual response to pharmacological treatments are two of the holy grails of Personalized Medicine. These tasks are challenging and require an integrated knowledge of the complex processes that regulate gene expression and, ultimately, protein functionality in vivo, the effects of mutations/polymorphisms and the different sources of interindividual phenotypic variability. A remarkable example of our advances in these challenging tasks is the highly polymorphic CYP2D6 gene, which encodes a cytochrome P450 enzyme involved in the metabolization of many of the most marketed drugs (including SARS-Cov-2 therapies such as hydroxychloroquine). Since the introduction of simple activity scores (AS) over 10 years ago, its ability to establish genotype-phenotype correlations on the drug metabolizing capacity of this enzyme in human population has provided lessons that will help to improve this type of score for this, and likely many other human genes and proteins. Multidisciplinary research emerges as the best approach to incorporate additional concepts to refine and improve such functional/activity scores for the CYP2D6 gene, as well as for many other human genes associated with simple and complex genetic diseases.