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

ERDF/Spanish Ministry of Science, Innovation RTI2018-096246-B-I00Junta de Andalucia P18-RT-2413ERDF/Counseling of Economic transformation, Industry, Knowledge, and Universities B-BIO-84-UGR2

A Dynamic Core in Human NQO1 Controls the Functional and Stability Effects of Ligand Binding and Their Communication across the Enzyme Dimer

Human NAD(P)H:quinone oxidoreductase 1 (NQO1) is a multi-functional protein whose alteration is associated with cancer, Parkinson’s and Alzheimer´s diseases. NQO1 displays a remarkable functional chemistry, capable of binding different functional ligands that modulate its activity, stability and interaction with proteins and nucleic acids. Our understanding of this functional chemistry is limited by the difficulty of obtaining structural and dynamic information on many of these states. Herein, we have used hydrogen/deuterium exchange monitored by mass spectrometry (HDXMS) to investigate the structural dynamics of NQO1 in three ligation states: without ligands (NQO1apo), with FAD (NQO1holo) and with FAD and the inhibitor dicoumarol (NQO1dic). We show that NQO1apo has a minimally stable folded core holding the protein dimer, with FAD and dicoumarol binding sites populating binding non-competent conformations. Binding of FAD significantly decreases protein dynamics and stabilizes the FAD and dicoumarol binding sites as well as the monomer:monomer interface. Dicoumarol binding further stabilizes all three functional sites, a result not previously anticipated by available crystallographic models. Our work provides an experimental perspective into the communication of stability effects through the NQO1 dimer, which is valuable for understanding at the molecular level the effects of disease-associated variants, post-translational modifications and ligand binding cooperativity in NQO1.This research was funded by the ERDF/Spanish Ministry of Science, Innovation and Universities—State Research Agency (Grant RTI2018-096246-B-I00, to A.L.P.), the Spanish Ministry of Economy and Competitiveness (Grant SAF2015-69796, to E.S.) and Junta de Andalucía (Grant P11-CTS-07187, to ALP). Access to an EU_FT–ICR_MS network installation was funded by the EU Horizon 2020 grant 731077. Additional support from Aula FUNCANIS-UGR, EU and MEYS CZ funds CZ.1.05/1.1.00/02.0109, LQ1604 and LM2015043 is gratefully acknowledged

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

This work was supported by the ERDF/Spanish Ministry of Science, Innovation and Universities-State Research Agency (Grant number RTI2018-096246-B-I00), Consejeria de Economia, Conocimiento, Empresas y Universidad, Junta de Andalucia (Grant number P18-RT-2413), ERDF/Counselling of Economic transformation, Industry, Knowledge and Universities (Grant B-BIO-84-UGR20), MCIN/AEI/10.13039/501100011033 (Grant number PID2019-103901GB-I00), Government of Aragon-FEDER (Grant number E35_20R). Financial support from Horizon 2020 EU_FT-ICR_MS project (Grant number 731077), EU/MEYS projects BioCeV (CZ.1.05/1.1.00/02.0109) and CIISB (Grant number LM2018127) is gratefully acknowledged. The funding sources had no role in study design, collection, analysis and interpretation of data, writing of the report; and in the decision to submit the article for publication. Funding for open access charge: Universidad de Granada/CBUA.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.ERDF/Spanish Ministry of Science, Innovation and Universities—State Research Agency RTI2018-096246-B-I00Junta de Andalucía P18-RT-2413ERDF/Counselling of Economic transformation, Industry, Knowledge and Universities B-BIO-84-UGR20MCIN/AEI/10.13039/501100011033 PID2019-103901GB-I00Government of Aragón-FEDER E35_20RHorizon 2020 EU_FT-ICR_MS 731077EU/MEYS projects BioCeV CZ.1.05/1.1.00/02.0109CIISB LM2018127Universidad de Granada/CBU

Effect of naturally-occurring mutations on the stability and function of cancer-associated NQO1: Comparison of experiments and computation

Recent advances in DNA sequencing technologies are revealing a large individual variability of the human genome. Our capacity to establish genotype-phenotype correlations in such large-scale is, however, limited. This task is particularly challenging due to the multifunctional nature of many proteins. Here we describe an extensive analysis of the stability and function of naturally-occurring variants (found in the COSMIC and gnomAD databases) of the cancer-associated human NAD(P)H:quinone oxidoreductase 1 (NQO1). First, we performed in silico saturation mutagenesis studies (>5,000 substitutions) aimed to identify regions in NQO1 important for stability and function. We then experimentally characterized twenty-two naturally-occurring variants in terms of protein levels during bacterial expression, solubility, thermal stability, and coenzyme binding. These studies showed a good overall correlation between experimental analysis and computational predictions; also the magnitude of the effects of the substitutions are similarly distributed in variants from the COSMIC and gnomAD databases. Outliers in these experimental-computational genotype-phenotype correlations remain, and we discuss these on the grounds and limitations of our approaches. Our work represents a further step to characterize the mutational landscape of NQO1 in the human genome and may help to improve high-throughput in silico tools for genotype-phenotype correlations in this multifunctional protein associated with disease.ERDF/Spanish Ministry of Science, Innovation and Universities-State Research AgencyJunta de Andalucia RTI 2018-096246-B-I00ERDF/Counseling of Economic transformation, Industry, Knowledge and Universities P18-RT-2413Comunidad Valenciana B-BIO-84-UGR20Novo Nordisk FoundationNovocure Limited CIAICO/2021/135 NNF18OC003395

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

ALP thanks Professors Jose Manuel Sanchez-Ruiz and Beatriz Ibarra-Molero (both from the University of Granada) for providing access and advice on their home-built software for electrostatic calculations. BR acknowledges kind hospitality and use of computational resources in the European Magnetic Resonance Center (CERM), Sesto Fiorentino (Florence), Italy. This work was supported by Spanish Ministry of Economy and Competitiveness and European ERDF Funds (MCIU/AEI/FEDER, EU) [RTI2018-097991-BI00 to JLN and RTI2018-096246-B-I00 to ALP]; FEDER/Junta de Andalucia-Consejeria de Transformacion Economica, Industria, Conocimiento y Universidades [Grant P18-RT-2413 to ALP]. Financial support from EU Horizon 2020 project EU FT-ICR MS (731077) as well as institutional (CZ.1.05/1.1.00/02.0109) and MS facility support (LM2018127 CIISB) are gratefully acknowledged. Funding for open charge: Universidad de Granada/CBUA.The phosphomimetic mutation S82D in the cancer-associated, FADdependent 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.Spanish GovernmentEuropean ERDF Funds (MCIU/AEI/FEDER, EU) RTI2018-097991-BI00 RTI2018-096246-B-I00FEDER/Junta de Andalucia-Consejeria de Transformacion Economica, Industria, Conocimiento y Universidades P18-RT-2413EU Horizon 2020 project EU FT-ICR MS 731077Universidad de Granada/CBUA CZ.1.05/1.1.00/02.0109 LM2018127 CIIS

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.ERDF/Spanish Ministry of Science, Innovation and Universities-State Research Agency RTI2018-096246-B-I00Junta de Andalucia P18-RT-2413ERDF/Counseling of Economic transformation, Industry, Knowledge and Universities B-BIO-84-UGR20Department of Science & Technology (India)Science Engineering Research Board (SERB), India MTR/2019/000392Horizon 2020 EU_FT-ICR_MS project 731077EU/MEYS projects BioCeV CZ.1.05/1.1.00/02.0109CIISB LM201812

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.STC Program of the National Science Foundation through BioXFEL (under agreement # 1231306)ABI Innovations award (NSF # 1565180), IIBR award (# 1943448)MCB award (1817862)National Institutes of Health award # R01GM095583US Department of Energy, Office of Science, Office of Basic Energy Sciences under contract # DE-AC02- 76SF00515Center for Structural Dynamics in Biology, NIH grant P41GM13968“Ayuda de Atracción y Retención de Talento Investigador” from the Community of Madrid, Spain (REF: 2019-T1/BMD-15552)ERDF/Spanish Ministry of Science, Innovation, and Universities—State Research Agency (grant RTI2018-096246-B- I00), Consejería de Economía, Conocimiento, Empresas, y Universidad, Junta de Andalucía (grant P18-RT-2413),ERDF/Counseling of Economic transformation, Industry, Knowledge, and Universities (grant B-BIO-84-UGR20

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.Comunidad Valenciana CIAICO/2021/135 AULA FUNCANIS-UGRERDF/Spanish Ministry of Science, Innovation, and Universities-State Research Agency RTI2018-096246-B-I00Junta de Andalucia P18-RT-2413 ERDF/ Counseling of Economic transformation, Industry, Knowledge, and Universities B-BIO-84-UGR2

NAD(P)H quinone oxidoreductase (NQO1): an enzyme which needs just enough mobility, in just the right places

NAD(P)H quinone oxidoreductase 1 (NQO1) catalyses the two electron reduction of quinones and a wide range of other organic compounds. Its physiological role is believed to be partly the reduction of free radical load in cells and the detoxification of xenobiotics. It also has non-enzymatic functions stabilising a number of cellular regulators including p53. Functionally, NQO1 is a homodimer with two active sites formed from residues from both polypeptide chains. Catalysis proceeds via a substituted enzyme mechanism involving a tightly bound FAD cofactor. Dicoumarol and some structurally related compounds act as competitive inhibitors of NQO1. There is some evidence for negative cooperativity in quinine oxidoreductases which is most likely to be mediated at least in part by alterations to the mobility of the protein. Human NQO1 is implicated in cancer. It is often over-expressed in cancer cells and as such is considered as a possible drug target. Interestingly, a common polymorphic form of human NQO1, p.P187S, is associated with an increased risk of several forms of cancer. This variant has much lower activity than the wild-type, primarily due to its substantially reduced affinity for FAD which results from lower stability. This lower stability results from inappropriate mobility of key parts of the protein. Thus, NQO1 relies on correct mobility for normal function, but inappropriate mobility results in dysfunction and may cause disease.This work was supported by the Junta de Andalucía [grant number P11-CTS-07187 (to A.L.P.)]

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 (approximate to 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.ERDF/Spanish Ministry of Science, Innovation and Universities-State Research Agency RTI2018-096246-B-I00Junta de Andalucia P18-RT-2413ERDF/Counseling of Economic transformation, Industry, Knowledge and Universities B-BIO-84-UGR20Government of Aragon-FEDER E35_20RDepartment of Science & Technology (India)Science Engineering Research Board (SERB), India MTR/2019/000392Horizon 2020 EPIC-XS project 82383EU/MEYS project BioCeV CZ.1.05/1.1.00/02.0109ERDF/Counseling of Economic transformation, Industry, Knowledge and Universities, Junta de Andalucia B-BIO-84-UGR20EU/MEYS project CIISB LM2018127MCIN/AEI PID2019-103901GB-I0