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1.
Biomol NMR Assign ; 14(2): 339-346, 2020 10.
Artigo em Inglês | MEDLINE | ID: mdl-32803496

RESUMO

The SARS-CoV-2 genome encodes for approximately 30 proteins. Within the international project COVID19-NMR, we distribute the spectroscopic analysis of the viral proteins and RNA. Here, we report NMR chemical shift assignments for the protein Nsp3b, a domain of Nsp3. The 217-kDa large Nsp3 protein contains multiple structurally independent, yet functionally related domains including the viral papain-like protease and Nsp3b, a macrodomain (MD). In general, the MDs of SARS-CoV and MERS-CoV were suggested to play a key role in viral replication by modulating the immune response of the host. The MDs are structurally conserved. They most likely remove ADP-ribose, a common posttranslational modification, from protein side chains. This de-ADP ribosylating function has potentially evolved to protect the virus from the anti-viral ADP-ribosylation catalyzed by poly-ADP-ribose polymerases (PARPs), which in turn are triggered by pathogen-associated sensing of the host immune system. This renders the SARS-CoV-2 Nsp3b a highly relevant drug target in the viral replication process. We here report the near-complete NMR backbone resonance assignment (1H, 13C, 15N) of the putative Nsp3b MD in its apo form and in complex with ADP-ribose. Furthermore, we derive the secondary structure of Nsp3b in solution. In addition, 15N-relaxation data suggest an ordered, rigid core of the MD structure. These data will provide a basis for NMR investigations targeted at obtaining small-molecule inhibitors interfering with the catalytic activity of Nsp3b.


Assuntos
Adenosina Difosfato Ribose/metabolismo , Apoproteínas/química , Betacoronavirus/metabolismo , Espectroscopia de Ressonância Magnética Nuclear de Carbono-13 , Isótopos de Nitrogênio/química , Espectroscopia de Prótons por Ressonância Magnética , Proteínas não Estruturais Virais/química , Sequência de Aminoácidos , Apoproteínas/metabolismo , Domínios Proteicos , Estrutura Secundária de Proteína , Proteínas não Estruturais Virais/metabolismo
2.
Nature ; 584(7822): 646-651, 2020 08.
Artigo em Inglês | MEDLINE | ID: mdl-32494015

RESUMO

Pannexin 1 (PANX1) is an ATP-permeable channel with critical roles in a variety of physiological functions such as blood pressure regulation1, apoptotic cell clearance2 and human oocyte development3. Here we present several structures of human PANX1 in a heptameric assembly at resolutions of up to 2.8 angström, including an apo state, a caspase-7-cleaved state and a carbenoxolone-bound state. We reveal a gating mechanism that involves two ion-conducting pathways. Under normal cellular conditions, the intracellular entry of the wide main pore is physically plugged by the C-terminal tail. Small anions are conducted through narrow tunnels in the intracellular domain. These tunnels connect to the main pore and are gated by a long linker between the N-terminal helix and the first transmembrane helix. During apoptosis, the C-terminal tail is cleaved by caspase, allowing the release of ATP through the main pore. We identified a carbenoxolone-binding site embraced by W74 in the extracellular entrance and a role for carbenoxolone as a channel blocker. We identified a gap-junction-like structure using a glycosylation-deficient mutant, N255A. Our studies provide a solid foundation for understanding the molecular mechanisms underlying the channel gating and inhibition of PANX1 and related large-pore channels.


Assuntos
Conexinas/química , Conexinas/metabolismo , Microscopia Crioeletrônica , Ativação do Canal Iônico , Proteínas do Tecido Nervoso/química , Proteínas do Tecido Nervoso/metabolismo , Técnicas de Patch-Clamp , Trifosfato de Adenosina/metabolismo , Animais , Apoproteínas/química , Apoproteínas/metabolismo , Apoproteínas/ultraestrutura , Apoptose , Sítios de Ligação/efeitos dos fármacos , Carbenoxolona/química , Carbenoxolona/metabolismo , Carbenoxolona/farmacologia , Caspase 7/metabolismo , Linhagem Celular , Conexinas/ultraestrutura , Junções Comunicantes , Glicosilação , Humanos , Ativação do Canal Iônico/efeitos dos fármacos , Modelos Moleculares , Mutação , Proteínas do Tecido Nervoso/ultraestrutura , Subunidades Proteicas/química , Subunidades Proteicas/metabolismo , Células Sf9
3.
Nat Commun ; 11(1): 3228, 2020 06 26.
Artigo em Inglês | MEDLINE | ID: mdl-32591529

RESUMO

Plasmodium falciparum (Pf) relies solely on the salvage pathway for its purine nucleotide requirements, making this pathway indispensable to the parasite. Purine nucleotide levels are regulated by anabolic processes and by nucleotidases that hydrolyse these metabolites into nucleosides. Certain apicomplexan parasites, including Pf, have an IMP-specific-nucleotidase 1 (ISN1). Here we show, by comprehensive substrate screening, that PfISN1 catalyzes the dephosphorylation of inosine monophosphate (IMP) and is allosterically activated by ATP. Crystal structures of tetrameric PfISN1 reveal complex rearrangements of domain organization tightly associated with catalysis. Immunofluorescence microscopy and expression of GFP-fused protein indicate cytosolic localization of PfISN1 and expression in asexual and gametocyte stages of the parasite. With earlier evidence on isn1 upregulation in female gametocytes, the structures reported in this study may contribute to initiate the design for possible transmission-blocking agents.


Assuntos
5'-Nucleotidase/química , 5'-Nucleotidase/metabolismo , Biocatálise , Plasmodium falciparum/enzimologia , Trifosfato de Adenosina/metabolismo , Animais , Apoproteínas/metabolismo , Sítios de Ligação , Concentração de Íons de Hidrogênio , Cinética , Magnésio/metabolismo , Camundongos Endogâmicos BALB C , Modelos Moleculares , Proteínas Mutantes/química , Domínios Proteicos , Estrutura Secundária de Proteína , Transporte Proteico , Proteínas de Protozoários/química , Proteínas de Protozoários/metabolismo , Especificidade por Substrato
4.
Biochim Biophys Acta Bioenerg ; 1861(10): 148240, 2020 10 01.
Artigo em Inglês | MEDLINE | ID: mdl-32531220

RESUMO

Complex I, NADH-ubiquinone oxidoreductase, is the first enzyme in the mitochondrial and bacterial aerobic respiratory chain. It pumps four protons through four transiently open pathways from the high pH, negative, N-side of the membrane to the positive, P-side driven by the exergonic transfer of electrons from NADH to a quinone. Three protons transfer through subunits descended from antiporters, while the fourth, E-channel is unique. The path through the E-channel is determined by a network analysis of hydrogen bonded pathways obtained by Monte Carlo sampling of protonation states, polar hydrogen orientation and water occupancy. Input coordinates are derived from molecular dynamics trajectories comparing oxidized, reduced (dihydro) and no menaquinone-8 (MQ). A complex proton transfer path from the N- to the P-side is found consisting of six clusters of highly connected hydrogen-bonded residues. The network connectivity depends on the presence of quinone and its redox state, supporting a role for this cofactor in coupling electron and proton transfers. The N-side is more organized with MQ-bound complex I facilitating proton entry, while the P-side is more connected in the apo-protein, facilitating proton exit. Subunit Nqo8 forms the core of the E channel; Nqo4 provides the N-side entry, Nqo7 and then Nqo10 join the pathway in the middle, while Nqo11 contributes to the P-side exit.


Assuntos
Complexo I de Transporte de Elétrons/química , Complexo I de Transporte de Elétrons/metabolismo , Prótons , Thermus thermophilus/enzimologia , Apoproteínas/química , Apoproteínas/metabolismo , Ligação de Hidrogênio , Simulação de Dinâmica Molecular , Conformação Proteica , Quinonas/metabolismo
5.
Nature ; 584(7820): 298-303, 2020 08.
Artigo em Inglês | MEDLINE | ID: mdl-32555460

RESUMO

Metabotropic γ-aminobutyric acid receptors (GABAB) are involved in the modulation of synaptic responses in the central nervous system and have been implicated in neuropsychological conditions that range from addiction to psychosis1. GABAB belongs to class C of the G-protein-coupled receptors, and its functional entity comprises an obligate heterodimer that is composed of the GB1 and GB2 subunits2. Each subunit possesses an extracellular Venus flytrap domain, which is connected to a canonical seven-transmembrane domain. Here we present four cryo-electron microscopy structures of the human full-length GB1-GB2 heterodimer: one structure of its inactive apo state, two intermediate agonist-bound forms and an active form in which the heterodimer is bound to an agonist and a positive allosteric modulator. The structures reveal substantial differences, which shed light on the complex motions that underlie the unique activation mechanism of GABAB. Our results show that agonist binding leads to the closure of the Venus flytrap domain of GB1, triggering a series of transitions, first rearranging and bringing the two transmembrane domains into close contact along transmembrane helix 6 and ultimately inducing conformational rearrangements in the GB2 transmembrane domain via a lever-like mechanism to initiate downstream signalling. This active state is stabilized by a positive allosteric modulator binding at the transmembrane dimerization interface.


Assuntos
Microscopia Crioeletrônica , Receptores de GABA-B/química , Receptores de GABA-B/ultraestrutura , Regulação Alostérica/efeitos dos fármacos , Apoproteínas/química , Apoproteínas/metabolismo , Apoproteínas/ultraestrutura , Sítios de Ligação/efeitos dos fármacos , Agonistas dos Receptores de GABA-B/química , Agonistas dos Receptores de GABA-B/metabolismo , Agonistas dos Receptores de GABA-B/farmacologia , Humanos , Modelos Moleculares , Domínios Proteicos/efeitos dos fármacos , Multimerização Proteica/efeitos dos fármacos , Receptores de GABA-B/metabolismo , Transdução de Sinais , Relação Estrutura-Atividade
6.
Biochim Biophys Acta Bioenerg ; 1861(8): 148214, 2020 08 01.
Artigo em Inglês | MEDLINE | ID: mdl-32360310

RESUMO

The structural features enabling carotenoid translocation between molecular entities in nature is poorly understood. Here, we present the three-dimensional X-ray structure of an expanded oligomeric state of the C-terminal domain homolog (CTDH) of the orange carotenoid protein, a key water-soluble protein in cyanobacterial photosynthetic photo-protection, at 2.9 Å resolution. This protein binds a canthaxanthin carotenoid ligand and undergoes structural reorganization at the dimeric level, which facilitates cargo uptake and delivery. The structure displays heterogeneity revealing the dynamic nature of its C-terminal tail (CTT). Molecular dynamics (MD) simulations based on the CTDH structures identified specific residues that govern the dimeric transition mechanism. Mutagenesis based on the crystal structure and these MD simulations then confirmed that these specific residues within the CTT are critical for carotenoid uptake, encapsulation and delivery processes. We present a mechanism that can be applied to other systems that require cargo uptake.


Assuntos
Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Carotenoides/metabolismo , Apoproteínas/química , Apoproteínas/metabolismo , Transporte Biológico , Cianobactérias/metabolismo , Simulação de Acoplamento Molecular , Simulação de Dinâmica Molecular , Domínios Proteicos , Multimerização Proteica , Estrutura Quaternária de Proteína
7.
Am J Physiol Heart Circ Physiol ; 318(5): H1296-H1307, 2020 05 01.
Artigo em Inglês | MEDLINE | ID: mdl-32302494

RESUMO

Haptoglobin (Hp) is the plasma protein that binds and clears cell-free hemoglobin (Hb), whereas apohemoglobin (apoHb, i.e., Hb devoid of heme) can bind heme. Therefore, the apoHb-Hp protein complex should facilitate holoHb-apoHb αß-dimer exchange and apoHb-heme intercalation. Thus, we hypothesized that apoHb-Hp could facilitate both Hb and heme clearance, which, if not alleviated, could have severe microcirculatory consequences. In this study, we characterized apoHb-Hp and Hb/heme ligand interactions and assessed their in vivo consequences. Hb exchange and heme binding with the apoHb-Hp complex was studied with transfer assays using size-exclusion high-performance liquid chromatography coupled with UV-visible spectrophotometry. Exchange/transfer experiments were conducted in guinea pigs dosed with Hb or heme-albumin followed by a challenge with equimolar amounts of apoHb-Hp. Finally, systemic and microcirculatory parameters were studied in hamsters instrumented with a dorsal window chamber via intravital microscopy. In vitro and in vivo Hb exchange and heme transfer experiments demonstrated proof-of-concept Hb/heme ligand transfer to apoHb-Hp. Dosing with the apoHb-Hp complex reversed Hb- and heme-induced systemic hypertension and microvascular vasoconstriction, reduced microvascular blood flow, and diminished functional capillary density. Therefore, this study highlights the apoHb-Hp complex as a novel therapeutic strategy to attenuate the adverse systemic and microvascular responses to intravascular Hb and heme exposure.NEW & NOTEWORTHY This study highlights the apoHb-Hp complex as a novel therapeutic strategy to attenuate the adverse systemic and microvascular responses to intravascular Hb and heme exposure. In vitro and in vivo Hb exchange and heme transfer experiments demonstrated proof-of-concept Hb/heme ligand transfer to apoHb-Hp. The apoHb-Hp complex reverses Hb- and heme-induced systemic hypertension and microvascular vasoconstriction, preserves microvascular blood flow, and functional capillary density. In summary, the unique properties of the apoHb-Hp complex prevent adverse systemic and microvascular responses to Hb and heme-albumin exposure and introduce a novel therapeutic approach to facilitate simultaneous removal of extracellular Hb and heme.


Assuntos
Apoproteínas/metabolismo , Haptoglobinas/metabolismo , Heme/metabolismo , Hemoglobinas/metabolismo , Hipertensão/sangue , Animais , Apoproteínas/sangue , Transfusão de Sangue/métodos , Cricetinae , Cobaias , Humanos , Hipertensão/fisiopatologia , Hipertensão/terapia , Masculino , Mesocricetus , Microcirculação , Ligação Proteica , Vasoconstrição
8.
Proc Natl Acad Sci U S A ; 117(18): 9857-9864, 2020 05 05.
Artigo em Inglês | MEDLINE | ID: mdl-32300017

RESUMO

Vitamin A has diverse biological functions and is essential for human survival at every point from embryogenesis to adulthood. Vitamin A and its derivatives have been used to treat human diseases including vision diseases, skin diseases, and cancer. Both insufficient and excessive vitamin A uptake are detrimental, but how its transport is regulated is poorly understood. STRA6 is a multitransmembrane domain cell-surface receptor and mediates vitamin A uptake from plasma retinol binding protein (RBP). STRA6 can mediate both cellular vitamin A influx and efflux, but what regulates these opposing activities is unknown. To answer this question, we purified and identified STRA6-associated proteins in a native mammalian cell type that takes up vitamin A through STRA6 using mass spectrometry. We found that the major protein repeatedly identified as STRA6-associated protein is calmodulin, consistent with the cryogenic electron microscopy (cryo-EM) study of zebrafish STRA6 associated with calmodulin. Using radioactivity-based, high-performance liquid chromatography (HPLC)-based and real-time fluorescence techniques, we found that calmodulin profoundly affects STRA6's vitamin A transport activity. Increased calcium/calmodulin promotes cellular vitamin A efflux and suppresses vitamin A influx through STRA6. Further mechanistic studies revealed that calmodulin enhances the binding of apo-RBP to STRA6, and this enhancement is much more pronounced for apo-RBP than holo-RBP. This study revealed that calmodulin regulates STRA6's vitamin A influx or efflux activity by modulating its preferential interaction with apo-RBP or holo-RBP. This molecular mechanism of regulating vitamin A transport may point to new directions to treat human diseases associated with insufficient or excessive vitamin A uptake.


Assuntos
Transporte Biológico/genética , Calmodulina/genética , Proteínas de Membrana/genética , Proteínas Plasmáticas de Ligação ao Retinol/genética , Vitamina A/metabolismo , Animais , Apoproteínas/genética , Apoproteínas/metabolismo , Cálcio/metabolismo , Bovinos , Linhagem Celular , Cromatografia Líquida de Alta Pressão , Microscopia Crioeletrônica , Humanos , Proteínas de Membrana/metabolismo , Ligação Proteica/genética , Receptores de Superfície Celular/genética , Proteínas Plasmáticas de Ligação ao Retinol/metabolismo , Vitamina A/genética , Peixe-Zebra/genética
9.
Molecules ; 25(3)2020 Feb 08.
Artigo em Inglês | MEDLINE | ID: mdl-32046362

RESUMO

A series of water-soluble copper(II) complexes based on 2,9-dimethyl-1,10-phenanthroline (dmphen) and mixed-ligands, containing PTA=O (1,3,5-triaza-7-phosphaadamantane-7-oxide) have been synthesized and fully characterized. Two types of complexes have been obtained, monocationic [Cu(NO3)(O-PTA=O)(dmphen)][PF6] (1), [Cu(Cl)(dmphen)2][PF6] (2), and neutral [Cu(NO3)2(dmphen)] (3). The solid-state structures of all complexes have been determined by single-crystal X-ray diffraction. Magnetic studies for the complex 1-3 indicated a very weak antiferromagnetic interaction between copper(II) ions in crystal lattice. Complexes were successfully evaluated for their cytotoxic activities on the normal human dermal fibroblast (NHDF) cell line and the antitumor activity using the human lung carcinoma (A549), epithelioid cervix carcinoma (HeLa), colon (LoVo), and breast adenocarcinoma (MCF-7) cell lines. Complexes 1 and 3 revealed lower toxicity to NHDF than A549 and HeLa cells, meanwhile compound 2 appeared to be more toxic to NHDF cell line in comparison to all cancer lines. Additionally, interactions between the complexes and human apo-transferrin (apo-Tf) using fluorescence and circular dichroism (CD) spectroscopy were also investigated. All compounds interacted with apo-transferrin, causing same changes of the protein conformation. Electrostatic interactions dominate in the 1/2 - apo- Tf systems and hydrophobic and ionic interactions in the case of 3.


Assuntos
Adamantano/química , Antineoplásicos/síntese química , Apoproteínas/química , Complexos de Coordenação/síntese química , Cobre/química , Fenantrolinas/química , Transferrina/química , Células A549 , Adamantano/análogos & derivados , Antineoplásicos/farmacologia , Apoproteínas/metabolismo , Cátions Bivalentes , Cátions Monovalentes , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Complexos de Coordenação/farmacologia , Fibroblastos/citologia , Fibroblastos/efeitos dos fármacos , Fibroblastos/metabolismo , Humanos , Concentração Inibidora 50 , Cinética , Células MCF-7 , Óxidos/química , Ligação Proteica , Termodinâmica , Transferrina/metabolismo
10.
Nature ; 579(7797): 152-157, 2020 03.
Artigo em Inglês | MEDLINE | ID: mdl-32076264

RESUMO

GPR52 is a class-A orphan G-protein-coupled receptor that is highly expressed in the brain and represents a promising therapeutic target for the treatment of Huntington's disease and several psychiatric disorders1,2. Pathological malfunction of GPR52 signalling occurs primarily through the heterotrimeric Gs protein2, but it is unclear how GPR52 and Gs couple for signal transduction and whether a native ligand or other activating input is required. Here we present the high-resolution structures of human GPR52 in three states: a ligand-free state, a Gs-coupled self-activation state and a potential allosteric ligand-bound state. Together, our structures reveal that extracellular loop 2 occupies the orthosteric binding pocket and operates as a built-in agonist, conferring an intrinsically high level of basal activity to GPR523. A fully active state is achieved when Gs is coupled to GPR52 in the absence of an external agonist. The receptor also features a side pocket for ligand binding. These insights into the structure and function of GPR52 could improve our understanding of other self-activated GPCRs, enable the identification of endogenous and tool ligands, and guide drug discovery efforts that target GPR52.


Assuntos
Receptores Acoplados a Proteínas-G/química , Receptores Acoplados a Proteínas-G/metabolismo , Regulação Alostérica , Sítio Alostérico , Motivos de Aminoácidos , Sequência de Aminoácidos , Apoproteínas/agonistas , Apoproteínas/química , Apoproteínas/metabolismo , Sítios de Ligação , Microscopia Crioeletrônica , Cristalografia por Raios X , Subunidades alfa Gs de Proteínas de Ligação ao GTP/química , Subunidades alfa Gs de Proteínas de Ligação ao GTP/metabolismo , Subunidades alfa Gs de Proteínas de Ligação ao GTP/ultraestrutura , Humanos , Ligantes , Modelos Moleculares , Receptores Acoplados a Proteínas-G/agonistas , Receptores Acoplados a Proteínas-G/ultraestrutura
11.
Nat Commun ; 11(1): 956, 2020 02 19.
Artigo em Inglês | MEDLINE | ID: mdl-32075969

RESUMO

Nuclear receptor (NR) transcription factors use a conserved activation function-2 (AF-2) helix 12 mechanism for agonist-induced coactivator interaction and NR transcriptional activation. In contrast, ligand-induced corepressor-dependent NR repression appears to occur through structurally diverse mechanisms. We report two crystal structures of peroxisome proliferator-activated receptor gamma (PPARγ) in an inverse agonist/corepressor-bound transcriptionally repressive conformation. Helix 12 is displaced from the solvent-exposed active conformation and occupies the orthosteric ligand-binding pocket enabled by a conformational change that doubles the pocket volume. Paramagnetic relaxation enhancement (PRE) NMR and chemical crosslinking mass spectrometry confirm the repressive helix 12 conformation. PRE NMR also defines the mechanism of action of the corepressor-selective inverse agonist T0070907, and reveals that apo-helix 12 exchanges between transcriptionally active and repressive conformations-supporting a fundamental hypothesis in the NR field that helix 12 exchanges between transcriptionally active and repressive conformations.


Assuntos
Benzamidas/metabolismo , Proteínas Correpressoras/metabolismo , PPAR gama/química , PPAR gama/metabolismo , Piridinas/metabolismo , Apoproteínas/química , Apoproteínas/metabolismo , Sítios de Ligação , Proteínas Correpressoras/química , Cristalografia por Raios X , Células HEK293 , Humanos , Ligantes , Espectroscopia de Ressonância Magnética , Mutação , Coativadores de Receptor Nuclear/química , Coativadores de Receptor Nuclear/metabolismo , PPAR gama/agonistas , PPAR gama/genética , Ligação Proteica , Conformação Proteica , Relação Estrutura-Atividade , Transcrição Genética
12.
Biochim Biophys Acta Bioenerg ; 1861(4): 148038, 2020 04 01.
Artigo em Inglês | MEDLINE | ID: mdl-31229568

RESUMO

Photosynthesis is a fundamental biological process involving the conversion of solar energy into chemical energy. The initial photochemical and photophysical events of photosynthesis are mediated by photosystem II (PSII) and photosystem I (PSI). Both PSII and PSI are multi-subunit supramolecular machineries composed of a core complex and a peripheral antenna system. The antenna system serves to capture light energy and transfer it to the core efficiently. Both PSII and PSI in the green lineage (plants and green algae) and PSI in red algae have an antenna system comprising a series of chlorophyll- and carotenoid-binding membrane proteins belonging to the light-harvesting complex (LHC) superfamily, including LHCII and LHCI. However, the antenna size and subunit composition vary considerably in the two photosystems from diverse organisms. On the basis of the plant and algal LHCII and LHCI structures that have been solved by X-ray crystallography and single-particle cryo-electron microscopy we review the detailed structural features and characteristic pigment properties of these LHCs in PSII and PSI. This article is part of a Special Issue entitled Light harvesting, edited by Dr. Roberta Croce.


Assuntos
Complexos de Proteínas Captadores de Luz/química , Sequência de Aminoácidos , Apoproteínas/química , Apoproteínas/metabolismo , Chlamydomonas reinhardtii/metabolismo , Clorofila/metabolismo , Complexos de Proteínas Captadores de Luz/metabolismo , Modelos Moleculares , Complexo de Proteína do Fotossistema I/metabolismo , Complexo de Proteína do Fotossistema II/química , Complexo de Proteína do Fotossistema II/metabolismo , Subunidades Proteicas/química , Subunidades Proteicas/metabolismo , Rodófitas/metabolismo
13.
Nature ; 574(7779): 581-585, 2019 10.
Artigo em Inglês | MEDLINE | ID: mdl-31645725

RESUMO

The tricarboxylic acid cycle intermediate succinate is involved in metabolic processes and plays a crucial role in the homeostasis of mitochondrial reactive oxygen species1. The receptor responsible for succinate signalling, SUCNR1 (also known as GPR91), is a member of the G-protein-coupled-receptor family2 and links succinate signalling to renin-induced hypertension, retinal angiogenesis and inflammation3-5. Because SUCNR1 senses succinate as an immunological danger signal6-which has relevance for diseases including ulcerative colitis, liver fibrosis7, diabetes and rheumatoid arthritis3,8-it is of interest as a therapeutic target. Here we report the high-resolution crystal structure of rat SUCNR1 in complex with an intracellular binding nanobody in the inactive conformation. Structure-based mutagenesis and radioligand-binding studies, in conjunction with molecular modelling, identified key residues for species-selective antagonist binding and enabled the determination of the high-resolution crystal structure of a humanized rat SUCNR1 in complex with a high-affinity, human-selective antagonist denoted NF-56-EJ40. We anticipate that these structural insights into the architecture of the succinate receptor and its antagonist selectivity will enable structure-based drug discovery and will further help to elucidate the function of SUCNR1 in vitro and in vivo.


Assuntos
Compostos de Bifenilo/química , Compostos de Bifenilo/farmacologia , Piperazinas/química , Piperazinas/farmacologia , Receptores Acoplados a Proteínas-G/antagonistas & inibidores , Receptores Acoplados a Proteínas-G/química , Animais , Apoproteínas/antagonistas & inibidores , Apoproteínas/química , Apoproteínas/metabolismo , Cristalografia por Raios X , Humanos , Modelos Moleculares , Ratos , Receptores Acoplados a Proteínas-G/metabolismo , Receptores Purinérgicos P2Y1/química , Transdução de Sinais , Anticorpos de Domínio Único/química , Especificidade da Espécie , Ácido Succínico/metabolismo
14.
Commun Biol ; 2: 351, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31583285

RESUMO

Photosynthetic organisms employ two different enzymes for the reduction of the C17 = C18 double bond of protochlorophyllide (Pchlide), yielding the chlorophyll precursor chlorophyllide. First, a nitrogenase-like, light-independent (dark-operative) Pchlide oxidoreductase and secondly, a light-dependent Pchlide oxidoreductase (LPOR). For the latter enzyme, despite decades of research, no structural information is available. Here, we use protein structure modelling, molecular dynamics (MD) simulations combined with multi-wavelength analytical ultracentrifugation (MWA-AUC) and small angle X-ray scattering (SAXS) experiments to derive a consensus model of the LPOR apoprotein and the substrate/cofactor/LPOR ternary complex. MWA-AUC and SAXS experiments independently demonstrate that the apoprotein is monomeric, while ternary complex formation induces dimerization. SAXS-guided modelling studies provide a full-length model of the apoprotein and suggest a tentative mode of dimerization for the LPOR ternary complex, supported by published cross-link constraints. Our study provides a first impression of the LPOR structural organization.


Assuntos
Cianobactérias/fisiologia , Oxirredutases atuantes sobre Doadores de Grupo CH-CH/metabolismo , Fotossíntese , Pigmentos Biológicos/metabolismo , Apoproteínas/química , Apoproteínas/metabolismo , Modelos Moleculares , Estrutura Molecular , Oxirredutases atuantes sobre Doadores de Grupo CH-CH/química , Pigmentos Biológicos/química , Multimerização Proteica , Relação Estrutura-Atividade
15.
Curr Protein Pept Sci ; 20(11): 1102-1111, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31553290

RESUMO

Calmodulin (CaM) is a highly conserved eukaryotic Ca2+ sensor protein that is able to bind a large variety of target sequences without a defined consensus sequence. The recognition of this diverse target set allows CaM to take part in the regulation of several vital cell functions. To fully understand the structural basis of the regulation functions of CaM, the investigation of complexes of CaM and its targets is essential. In this minireview we give an outline of the different types of CaM - peptide complexes with 3D structure determined, also providing an overview of recently determined structures. We discuss factors defining the orientations of peptides within the complexes, as well as roles of anchoring residues. The emphasis is on complexes where multiple binding modes were found.


Assuntos
Calmodulina/química , Calmodulina/metabolismo , Peptídeos/metabolismo , Animais , Apoproteínas/química , Apoproteínas/metabolismo , Cálcio/metabolismo , Humanos , Ligação Proteica
16.
Nucleic Acids Res ; 47(16): 8888-8898, 2019 09 19.
Artigo em Inglês | MEDLINE | ID: mdl-31372631

RESUMO

DNA mismatch repair (MMR) corrects mismatches, small insertions and deletions in DNA during DNA replication. While scanning for mismatches, dimers of MutS embrace the DNA helix with their lever and clamp domains. Previous studies indicated generic flexibility of the lever and clamp domains of MutS prior to DNA binding, but whether this was important for MutS function was unknown. Here, we present a novel crystal structure of DNA-free Escherichia coli MutS. In this apo-structure, the clamp domains are repositioned due to kinking at specific sites in the coiled-coil region in the lever domains, suggesting a defined hinge point. We made mutations at the coiled-coil hinge point. The mutants made to disrupt the helical fold at the kink site diminish DNA binding, whereas those made to increase stability of coiled-coil result in stronger DNA binding. These data suggest that the site-specific kinking of the coiled-coil in the lever domain is important for loading of this ABC-ATPase on DNA.


Assuntos
Apoproteínas/química , DNA Bacteriano/química , Proteínas de Escherichia coli/química , Escherichia coli/genética , Proteína MutS de Ligação de DNA com Erro de Pareamento/química , Sequência de Aminoácidos , Apoproteínas/genética , Apoproteínas/metabolismo , Sítios de Ligação , Clonagem Molecular , Cristalografia por Raios X , DNA Bacteriano/genética , DNA Bacteriano/metabolismo , Escherichia coli/metabolismo , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Expressão Gênica , Vetores Genéticos/química , Vetores Genéticos/metabolismo , Cinética , Modelos Moleculares , Proteína MutS de Ligação de DNA com Erro de Pareamento/genética , Proteína MutS de Ligação de DNA com Erro de Pareamento/metabolismo , Mutagênese Sítio-Dirigida , Mutação , Ligação Proteica , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha beta , Domínios e Motivos de Interação entre Proteínas , Multimerização Proteica , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Alinhamento de Sequência , Relação Estrutura-Atividade
17.
Life Sci ; 233: 116710, 2019 Sep 15.
Artigo em Inglês | MEDLINE | ID: mdl-31369762

RESUMO

AIMS: The naturally occurring compound curcumin has been proposed for a number of pharmacological applications. In spite of the promising chemotherapeutic properties of the molecule, the use of curcumin has been largely limited by its chemical instability in water. In this work, we propose the use of water soluble proteins to overcome this issue in perspective applications to photodynamic therapy of tumors. MATERIALS AND METHODS: Curcumin was bound to bovine serum albumin and its photophysical properties was studied as well as its effect on cell viability after light exposure through MTT assay and confocal imaging. KEY FINDINGS: Bovine serum albumin binds curcumin with moderate affinity and solubilizes the hydrophobic compound preserving its photophysical properties for several hours. Cell viability assays demonstrate that when bound to serum albumin, curcumin is an effective photosensitizer for HeLa cells, with better performance than curcumin alone. Confocal fluorescence imaging reveals that when curcumin is delivered alone, it preferentially associates with mitochondria, whereas curcumin bound to bovine serum albumin is found in additional locations within the cell, a fact that may be related to the higher phototoxicity observed in this case. SIGNIFICANCE: The higher bioavailability of the photosensitizing compound curcumin when bound to serum albumin may be exploited to increase the efficiency of the drug in photodynamic therapy of tumors.


Assuntos
Apoproteínas/metabolismo , Apoptose/efeitos dos fármacos , Curcumina/farmacologia , Sistemas de Liberação de Medicamentos , Mioglobina/metabolismo , Fotoquimioterapia , Fármacos Fotossensibilizantes/farmacologia , Soroalbumina Bovina/metabolismo , Animais , Apoproteínas/química , Apoptose/efeitos da radiação , Bovinos , Sobrevivência Celular , Curcumina/química , Células HeLa , Cavalos , Humanos , Mioglobina/química , Fármacos Fotossensibilizantes/química , Soroalbumina Bovina/química
18.
Sci Adv ; 5(7): eaaw8478, 2019 07.
Artigo em Inglês | MEDLINE | ID: mdl-31392273

RESUMO

The mechanisms underlying the biogenesis of the structurally unique, binuclear Cu1.5+•Cu1.5+ redox center (CuA) on subunit II (CoxB) of cytochrome oxidases have been a long-standing mystery. Here, we reconstituted the CoxB•CuA center in vitro from apo-CoxB and the holo-forms of the copper transfer chaperones ScoI and PcuC. A previously unknown, highly stable ScoI•Cu2+•CoxB complex was shown to be rapidly formed as the first intermediate in the pathway. Moreover, our structural data revealed that PcuC has two copper-binding sites, one each for Cu1+ and Cu2+, and that only PcuC•Cu1+•Cu2+ can release CoxB•Cu2+ from the ScoI•Cu2+•CoxB complex. The CoxB•CuA center was then formed quantitatively by transfer of Cu1+ from a second equivalent of PcuC•Cu1+•Cu2+ to CoxB•Cu2+. This metalation pathway is consistent with all available in vivo data and identifies the sources of the Cu ions required for CuA center formation and the order of their delivery to CoxB.


Assuntos
Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Cobre/metabolismo , Complexo IV da Cadeia de Transporte de Elétrons/química , Complexo IV da Cadeia de Transporte de Elétrons/metabolismo , Metalochaperonas/química , Metalochaperonas/metabolismo , Apoproteínas/metabolismo , Sítios de Ligação , Bradyrhizobium/metabolismo , Cristalografia por Raios X , Modelos Biológicos , Oxirredução , Domínios Proteicos , Relação Estrutura-Atividade
19.
Proc Natl Acad Sci U S A ; 116(32): 15802-15810, 2019 08 06.
Artigo em Inglês | MEDLINE | ID: mdl-31337676

RESUMO

The active site (H-cluster) of [FeFe]-hydrogenases is a blueprint for the design of a biologically inspired H2-producing catalyst. The maturation process describes the preassembly and uptake of the unique [2FeH] cluster into apo-hydrogenase, which is to date not fully understood. In this study, we targeted individual amino acids by site-directed mutagenesis in the [FeFe]-hydrogenase CpI of Clostridium pasteurianum to reveal the final steps of H-cluster maturation occurring within apo-hydrogenase. We identified putative key positions for cofactor uptake and the subsequent structural reorganization that stabilizes the [2FeH] cofactor in its functional coordination sphere. Our results suggest that functional integration of the negatively charged [2FeH] precursor requires the positive charges and individual structural features of the 2 basic residues of arginine 449 and lysine 358, which mark the entrance and terminus of the maturation channel, respectively. The results obtained for 5 glycine-to-histidine exchange variants within a flexible loop region provide compelling evidence that the glycine residues function as hinge positions in the refolding process, which closes the secondary ligand sphere of the [2FeH] cofactor and the maturation channel. The conserved structural motifs investigated here shed light on the interplay between the secondary ligand sphere and catalytic cofactor.


Assuntos
Hidrogenase/metabolismo , Ferro/metabolismo , Apoproteínas/química , Apoproteínas/metabolismo , Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Clostridium/enzimologia , Eletroquímica , Holoenzimas/química , Holoenzimas/metabolismo , Hidrogênio/metabolismo , Hidrogenase/química , Modelos Moleculares , Espectroscopia de Infravermelho com Transformada de Fourier
20.
Nature ; 572(7769): 347-351, 2019 08.
Artigo em Inglês | MEDLINE | ID: mdl-31278385

RESUMO

The high-conductance intracellular calcium (Ca2+) channel RyR2 is essential for the coupling of excitation and contraction in cardiac muscle. Among various modulators, calmodulin (CaM) regulates RyR2 in a Ca2+-dependent manner. Here we reveal the regulatory mechanism by which porcine RyR2 is modulated by human CaM through the structural determination of RyR2 under eight conditions. Apo-CaM and Ca2+-CaM bind to distinct but overlapping sites in an elongated cleft formed by the handle, helical and central domains. The shift in CaM-binding sites on RyR2 is controlled by Ca2+ binding to CaM, rather than to RyR2. Ca2+-CaM induces rotations and intradomain shifts of individual central domains, resulting in pore closure of the PCB95 and Ca2+-activated channel. By contrast, the pore of the ATP, caffeine and Ca2+-activated channel remains open in the presence of Ca2+-CaM, which suggests that Ca2+-CaM is one of the many competing modulators of RyR2 gating.


Assuntos
Calmodulina/metabolismo , Canal de Liberação de Cálcio do Receptor de Rianodina/metabolismo , Trifosfato de Adenosina/metabolismo , Animais , Apoproteínas/metabolismo , Sítios de Ligação , Cafeína/metabolismo , Cálcio/metabolismo , Microscopia Crioeletrônica , Humanos , Modelos Moleculares , Reprodutibilidade dos Testes , Canal de Liberação de Cálcio do Receptor de Rianodina/química , Suínos
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