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

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

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\u2192G 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 ( 4820 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

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

JLP-G and ALP were supported by the ERDF/Spanish Ministry of Science, Innovation and Universities-State Research Agency (Grant RTI2018-096246-B-I00) and Consejeria de Economia, Conocimiento, Empresas y Universidad, Junta de Andalucia (Grants P11-CTS-7187 and P18-RT-2413) . NM-T was supported by Aula FUNCANIS-UGR. ES was supported by the ERDF/Spanish Ministry of Science, Innovation and Universities-State Research Agency (Grant SAF2015-69796) . Access to an EU_FT-ICR_MS network installation was funded by the EU Horizon 2020 grant 731077. EA-C and MM were supported by the Spanish Ministry of Science and Innovation-State Research Agency (Grant PID2019-103901 GB-I00) and Gobierno de Aragon-FEDER (Grant E35_20R) . Support of the BioCeV center (CZ.1.05/1.1.00/02.0109) and the CMS/CIISB facility (MEYS CZ-LM2018127) is also gratefully acknowledged. ANN was supported by grants BT/PR26099/BID/7/811/2017 from Department of Biotechnology (DBT, India) and MTR/2019/000392 from Science, Engineering and Research Board (SERB, India) .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 longrange 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.ERDF/Spanish Ministry of Science, Innovation and Universities-State Research Agency RTI2018-096246-B-I00- SAF2015-69796Junta de Andalucia P11-CTS-7187- P18-RT-2413Aula FUNCANIS-UGREuropean Commission 731077Spanish Ministry of Science and Innovation-State Research Agency PID2019-103901 GB-I00Gobierno de Aragon-FEDER E35_20RBioCeV center CZ.1.05/1.1.00/02.0109CMS/CIISB facility MEYS CZ-LM2018127Department of Biotechnology (DBT) India BT/PR26099/BID/7/811/2017Science, Engineering and Research Board (SERB, India) MTR/2019/00039

In-solution structure and oligomerization of human histone deacetylase 6 – an integrative approach

Human histone deacetylase 6 (HDAC6) is a structurally unique, multido-main protein implicated in a variety of physiological processes includingcytoskeletal remodelling and the maintenance of cellular homeostasis. Ourcurrent understanding of the HDAC6 structure is limited to isolateddomains, and a holistic picture of the full-length protein structure, includ-ing possible domain interactions, is missing. Here, we used an integrativestructural biology approach to build a solution model of HDAC6 by com-bining experimental data from several orthogonal biophysical techniquescomplemented by molecular modelling. We show that HDAC6 is bestdescribed as a mosaic of folded and intrinsically disordered domains thatin-solution adopts an ensemble of conformations without any stable inter-actions between structured domains. Furthermore, HDAC6 forms dimers/higher oligomers in a concentration-dependent manner, and its oligomer-ization is mediated via the positively charged N-terminal microtubule-binding domain. Our findings provide the first insights into the structure offull-length human HDAC6 and can be used as a basis for further researchinto structure function and physiological studies of this unique deacetylase

Human Stress-inducible Hsp70 Has a High Propensity to Form ATP-dependent Antiparallel Dimers That Are Differentially Regulated by Cochaperone Binding

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

15 pags., 6 figs.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.Access to the Structural Mass Spectrometry core facility—BioCeV was funded by the CIISB, Instruct-CZCentre, supported by MEYS CR (LM2023042). Euro-pean Regional Development Fund-Project “UPCIISB” (No. CZ.02.1.01/0.0/0.0/18_046/0015974) andEPIC-XS H2020 (823839) are gratefully acknowledged.The research of JLP-G, AG-M, and ALP was sup-ported by the ERDF/Spanish Ministry of Science,Innovation and Universities—State Research Agency(Grant RTI2018-096246-B-I00), Consejerıa de Economıa, Conocimiento, Empresas y Universidad, Junta deAndalucıa (Grant P18-RT-2413) and ERDF/Counsel-ing of Economic transformation, Industry, Knowledgeand Universities (Grant B-BIO-84-UGR20Peer reviewe