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1.
Signal Transduct Target Ther ; 5(1): 220, 2020 10 06.
Artigo em Inglês | MEDLINE | ID: mdl-33024075
2.
Nat Commun ; 11(1): 5081, 2020 10 08.
Artigo em Inglês | MEDLINE | ID: mdl-33033236

RESUMO

Diatom is an important group of marine algae and contributes to around 20% of the global photosynthetic carbon fixation. Photosystem I (PSI) of diatoms is associated with a large number of fucoxanthin-chlorophyll a/c proteins (FCPIs). We report the structure of PSI-FCPI from a diatom Chaetoceros gracilis at 2.38 Å resolution by single-particle cryo-electron microscopy. PSI-FCPI is a monomeric supercomplex consisting of 12 core and 24 antenna subunits (FCPIs), and 326 chlorophylls a, 34 chlorophylls c, 102 fucoxanthins, 35 diadinoxanthins, 18 ß-carotenes and some electron transfer cofactors. Two subunits designated PsaR and PsaS were found in the core, whereas several subunits were lost. The large number of pigments constitute a unique and huge network ensuring efficient energy harvesting, transfer and dissipation. These results provide a firm structural basis for unraveling the mechanisms of light-energy harvesting, transfer and quenching in the diatom PSI-FCPI, and also important clues to evolutionary changes of PSI-LHCI.


Assuntos
Proteínas de Ligação à Clorofila/química , Diatomáceas/metabolismo , Transferência de Energia , Complexo de Proteína do Fotossistema I/química , Complexos de Proteínas Captadores de Luz/química , Complexos de Proteínas Captadores de Luz/metabolismo , Pigmentos Biológicos/química , Pigmentos Biológicos/metabolismo , Subunidades Proteicas/química
3.
PLoS One ; 15(8): e0237711, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32810182

RESUMO

Alleles at the Glu-1 loci play important roles in the functional properties of wheat flour. The effects of various high-molecular-weight glutenin subunit (HMW-GS) compositions on quality traits and bread-making properties were evaluated using 235 doubled haploid lines (DHs). The experiment was conducted in a split plot design with two water regimes as the main plot treatment, and DH lines as the subplot treatments. Results showed that the presence of subunit pair 5+10 at the Glu-D1 locus, either alone or in combination with others, appears to provide an improvement in quality and bread-making properties. At the Glu-A1 locus, subunit 1 produced a higher Zeleny sedimentation value (Zel) and stretch area (SA) than subunit 2* when subunits 14+15 and 5+10 were expressed at the Glu-B1 and Glu-D1 loci, and 2* had a positive effect on the maximum dough resistance (Rmax) when subunits 14+15 and 5'+12 were expressed at the Glu-B1 and Glu-D1 loci, respectively. Given subunit 1 at the Glu-A1 locus and 5'+12 at the Glu-D1 locus, the effects of Glu-B1 subunits 14+15 on the tractility (Tra), dough stability time (ST), and dough development time (DT) under the well-watered regime were significantly higher than those of Glu-B1 subunits 13+16. However, 13+16 had a positive effect on SA under the rain-fed regime when subunits 2* and 5+10 were expressed at the Glu-A1 and Glu-D1 loci, respectively. Multiple comparisons analysis revealed that the Zel and Rmax of the six subunits and eight HMW-GS compositions were stable under different water regimes. Overall, subunit compositions 1, 13+16 and 5+10 and 1, 14+15 and 5+10 had higher values for quality traits and bread-baking properties under the two water regimes. These results could play a positive guiding role in selecting and popularizing varieties suitable for production and cultivation in local areas.


Assuntos
Qualidade dos Alimentos , Glutens/genética , Melhoramento Vegetal , Subunidades Proteicas/genética , Triticum/química , Irrigação Agrícola/métodos , Pão/normas , China , Farinha/normas , Genes de Plantas/genética , Loci Gênicos , Glutens/metabolismo , Haploidia , Peso Molecular , Subunidades Proteicas/química , Triticum/genética , Triticum/crescimento & desenvolvimento
4.
Nat Commun ; 11(1): 4245, 2020 08 25.
Artigo em Inglês | MEDLINE | ID: mdl-32843629

RESUMO

Diheme-containing succinate:menaquinone oxidoreductases (Sdh) are widespread in Gram-positive bacteria but little is known about the catalytic mechanisms they employ for succinate oxidation by menaquinone. Here, we present the 2.8 Å cryo-electron microscopy structure of a Mycobacterium smegmatis Sdh, which forms a trimer. We identified the membrane-anchored SdhF as a subunit of the complex. The 3 kDa SdhF forms a single transmembrane helix and this helix plays a role in blocking the canonically proximal quinone-binding site. We also identified two distal quinone-binding sites with bound quinones. One distal binding site is formed by neighboring subunits of the complex. Our structure further reveals the electron/proton transfer pathway for succinate oxidation by menaquinone. Moreover, this study provides further structural insights into the physiological significance of a trimeric respiratory complex II. The structure of the menaquinone binding site could provide a framework for the development of Sdh-selective anti-mycobacterial drugs.


Assuntos
Proteínas de Bactérias/química , Mycobacterium smegmatis/enzimologia , Succinato Desidrogenase/química , Proteínas de Bactérias/metabolismo , Sítios de Ligação , Catálise , Microscopia Crioeletrônica , Transporte de Elétrons , Modelos Moleculares , Complexos Multienzimáticos/química , Complexos Multienzimáticos/genética , Complexos Multienzimáticos/metabolismo , Mycobacterium smegmatis/química , Oxirredução , Subunidades Proteicas/química , Subunidades Proteicas/metabolismo , Relação Estrutura-Atividade , Succinato Desidrogenase/metabolismo , Ácido Succínico/metabolismo , Vitamina K 2/metabolismo
5.
Proc Natl Acad Sci U S A ; 117(28): 16373-16382, 2020 07 14.
Artigo em Inglês | MEDLINE | ID: mdl-32601233

RESUMO

In photosynthetic reaction centers from purple bacteria (PbRC) and the water-oxidizing enzyme, photosystem II (PSII), charge separation occurs along one of the two symmetrical electron-transfer branches. Here we report the microscopic origin of the unidirectional charge separation, fully considering electron-hole interaction, electronic coupling of the pigments, and electrostatic interaction with the polarizable entire protein environments. The electronic coupling between the pair of bacteriochlorophylls is large in PbRC, forming a delocalized excited state with the lowest excitation energy (i.e., the special pair). The charge-separated state in the active branch is stabilized by uncharged polar residues in the transmembrane region and charged residues on the cytochrome c 2 binding surface. In contrast, the accessory chlorophyll in the D1 protein (ChlD1) has the lowest excitation energy in PSII. The charge-separated state involves ChlD1 •+ and is stabilized predominantly by charged residues near the Mn4CaO5 cluster and the proceeding proton-transfer pathway. It seems likely that the acquirement of water-splitting ability makes ChlD1 the initial electron donor in PSII.


Assuntos
Elétrons , Complexo de Proteínas do Centro de Reação Fotossintética/metabolismo , Água/metabolismo , Aminoácidos , Bacterioclorofilas/química , Bacterioclorofilas/metabolismo , Transporte de Elétrons , Oxigênio/metabolismo , Complexo de Proteínas do Centro de Reação Fotossintética/química , Complexo de Proteína do Fotossistema II/química , Complexo de Proteína do Fotossistema II/metabolismo , Subunidades Proteicas/química , Subunidades Proteicas/metabolismo , Proteobactérias/metabolismo , Água/química
6.
Proc Natl Acad Sci U S A ; 117(29): 17011-17018, 2020 07 21.
Artigo em Inglês | MEDLINE | ID: mdl-32636271

RESUMO

Few antibiotics are effective against Acinetobacter baumannii, one of the most successful pathogens responsible for hospital-acquired infections. Resistance to chlorhexidine, an antiseptic widely used to combat A. baumannii, is effected through the proteobacterial antimicrobial compound efflux (PACE) family. The prototype membrane protein of this family, AceI (Acinetobacter chlorhexidine efflux protein I), is encoded for by the aceI gene and is under the transcriptional control of AceR (Acinetobacter chlorhexidine efflux protein regulator), a LysR-type transcriptional regulator (LTTR) protein. Here we use native mass spectrometry to probe the response of AceI and AceR to chlorhexidine assault. Specifically, we show that AceI forms dimers at high pH, and that binding to chlorhexidine facilitates the functional form of the protein. Changes in the oligomerization of AceR to enable interaction between RNA polymerase and promoter DNA were also observed following chlorhexidine assault. Taken together, these results provide insight into the assembly of PACE family transporters and their regulation via LTTR proteins on drug recognition and suggest potential routes for intervention.


Assuntos
Acinetobacter baumannii , Antibacterianos , Proteínas de Bactérias , Clorexidina , Proteínas de Membrana Transportadoras , Acinetobacter baumannii/química , Acinetobacter baumannii/enzimologia , Antibacterianos/química , Antibacterianos/metabolismo , Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Clorexidina/química , Clorexidina/metabolismo , RNA Polimerases Dirigidas por DNA/química , RNA Polimerases Dirigidas por DNA/metabolismo , Resistência Microbiana a Medicamentos , Concentração de Íons de Hidrogênio , Espectrometria de Massas , Proteínas de Membrana Transportadoras/química , Proteínas de Membrana Transportadoras/metabolismo , Ligação Proteica , Multimerização Proteica , Subunidades Proteicas/química , Subunidades Proteicas/metabolismo
7.
Nat Commun ; 11(1): 3583, 2020 07 17.
Artigo em Inglês | MEDLINE | ID: mdl-32681005

RESUMO

The phosphatases PP1 and PP2A are responsible for the majority of dephosphorylation reactions on phosphoserine (pSer) and phosphothreonine (pThr), and are involved in virtually all cellular processes and numerous diseases. The catalytic subunits exist in cells in form of holoenzymes, which impart substrate specificity. The contribution of the catalytic subunits to the recognition of substrates is unclear. By developing a phosphopeptide library approach and a phosphoproteomic assay, we demonstrate that the specificity of PP1 and PP2A holoenzymes towards pThr and of PP1 for basic motifs adjacent to the phosphorylation site are due to intrinsic properties of the catalytic subunits. Thus, we dissect this amino acid specificity of the catalytic subunits from the contribution of regulatory proteins. Furthermore, our approach enables discovering a role for PP1 as regulator of the GRB-associated-binding protein 2 (GAB2)/14-3-3 complex. Beyond this, we expect that this approach is broadly applicable to detect enzyme-substrate recognition preferences.


Assuntos
Proteína Fosfatase 1/química , Proteína Fosfatase 1/metabolismo , Proteína Fosfatase 2/química , Proteína Fosfatase 2/metabolismo , Proteínas 14-3-3/genética , Proteínas 14-3-3/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Motivos de Aminoácidos , Domínio Catalítico , Holoenzimas/química , Holoenzimas/genética , Holoenzimas/metabolismo , Humanos , Fosforilação , Ligação Proteica , Engenharia de Proteínas , Proteína Fosfatase 1/genética , Proteína Fosfatase 2/genética , Subunidades Proteicas/química , Subunidades Proteicas/genética , Subunidades Proteicas/metabolismo , Especificidade por Substrato
8.
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
9.
Nature ; 584(7820): 310-314, 2020 08.
Artigo em Inglês | MEDLINE | ID: mdl-32580208

RESUMO

Stimulation of the metabotropic GABAB receptor by γ-aminobutyric acid (GABA) results in prolonged inhibition of neurotransmission, which is central to brain physiology1. GABAB belongs to family C of the G-protein-coupled receptors, which operate as dimers to transform synaptic neurotransmitter signals into a cellular response through the binding and activation of heterotrimeric G proteins2,3. However, GABAB is unique in its function as an obligate heterodimer in which agonist binding and G-protein activation take place on distinct subunits4,5. Here we present cryo-electron microscopy structures of heterodimeric and homodimeric full-length GABAB receptors. Complemented by cellular signalling assays and atomistic simulations, these structures reveal that extracellular loop 2 (ECL2) of GABAB has an essential role in relaying structural transitions by ordering the linker that connects the extracellular ligand-binding domain to the transmembrane region. Furthermore, the ECL2 of each of the subunits of GABAB caps and interacts with the hydrophilic head of a phospholipid that occupies the extracellular half of the transmembrane domain, thereby providing a potentially crucial link between ligand binding and the receptor core that engages G proteins. These results provide a starting framework through which to decipher the mechanistic modes of signal transduction mediated by GABAB dimers, and have important implications for rational drug design that targets these receptors.


Assuntos
Microscopia Crioeletrônica , Receptores de GABA-B/química , Receptores de GABA-B/ultraestrutura , Sítios de Ligação , Membrana Celular/metabolismo , Antagonistas de Receptores de GABA-B/química , Antagonistas de Receptores de GABA-B/metabolismo , Humanos , Interações Hidrofóbicas e Hidrofílicas , Ligantes , Modelos Moleculares , Fosfolipídeos/química , Fosfolipídeos/metabolismo , Domínios Proteicos , Multimerização Proteica , Subunidades Proteicas/química , Subunidades Proteicas/metabolismo , Receptores de GABA-B/metabolismo , Receptores de Glutamato/química , Receptores de Glutamato/metabolismo , Transdução de Sinais , Relação Estrutura-Atividade
10.
Nat Commun ; 11(1): 2799, 2020 06 03.
Artigo em Inglês | MEDLINE | ID: mdl-32493979

RESUMO

Small molecule polyamines are abundant in all life forms and participate in diverse aspects of cell growth and differentiation. Spermidine/spermine acetyltransferase (SAT1) is the rate-limiting enzyme in polyamine catabolism and a primary genetic risk factor for suicidality. Here, using genome-wide screening, we find that SAT1 selectively controls nicotinic acetylcholine receptor (nAChR) biogenesis. SAT1 specifically augments assembly of nAChRs containing α7 or α4ß2, but not α6 subunits. Polyamines are classically studied as regulators of ion channel gating that engage the nAChR channel pore. In contrast, we find polyamine effects on assembly involve the nAChR cytosolic loop. Neurological studies link brain polyamines with neurodegenerative conditions. Our pharmacological and transgenic animal studies find that reducing polyamines enhances cortical neuron nAChR expression and augments nicotine-mediated neuroprotection. Taken together, we describe a most unexpected role for polyamines in regulating ion channel assembly, which provides a new avenue for nAChR neuropharmacology.


Assuntos
Canais Iônicos/metabolismo , Poliaminas/metabolismo , Receptores Nicotínicos/metabolismo , Acetiltransferases , Animais , Biocatálise , DNA Complementar/genética , Elementos Facilitadores Genéticos/genética , Fluorescência , Genoma Humano , Células HEK293 , Humanos , Ativação do Canal Iônico , Camundongos , Neurônios/metabolismo , Neuroproteção/efeitos dos fármacos , Estrutura Secundária de Proteína , Subunidades Proteicas/química , Subunidades Proteicas/metabolismo , Ratos , Receptores Nicotínicos/química
11.
Nat Commun ; 11(1): 2866, 2020 06 08.
Artigo em Inglês | MEDLINE | ID: mdl-32513959

RESUMO

The Cullin 5 (CUL5) Ring E3 ligase uses adaptors Elongins B and C (ELOB/C) to bind different SOCS-box-containing substrate receptors, determining the substrate specificity of the ligase. The 18-member ankyrin and SOCS box (ASB) family is the largest substrate receptor family. Here we report cryo-EM data for the substrate, creatine kinase (CKB) bound to ASB9-ELOB/C, and for full-length CUL5 bound to the RING protein, RBX2, which binds various E2s. To date, no full structures are available either for a substrate-bound ASB nor for CUL5. Hydrogen-deuterium exchange (HDX-MS) mapped onto a full structural model of the ligase revealed long-range allostery extending from the substrate through CUL5. We propose a revised allosteric mechanism for how CUL-E3 ligases function. ASB9 and CUL5 behave as rigid rods, connected through a hinge provided by ELOB/C transmitting long-range allosteric crosstalk from the substrate through CUL5 to the RBX2 flexible linker.


Assuntos
Creatina Quinase/metabolismo , Microscopia Crioeletrônica , Elonguina/metabolismo , Proteínas Supressoras da Sinalização de Citocina/química , Proteínas Supressoras da Sinalização de Citocina/metabolismo , Regulação Alostérica , Creatina Quinase/ultraestrutura , Proteínas Culina/química , Proteínas Culina/metabolismo , Elonguina/ultraestrutura , Humanos , Modelos Moleculares , Ligação Proteica , Subunidades Proteicas/química , Subunidades Proteicas/metabolismo , Relação Estrutura-Atividade , Especificidade por Substrato , Proteínas Supressoras da Sinalização de Citocina/ultraestrutura , Ubiquitina-Proteína Ligases/química , Ubiquitina-Proteína Ligases/metabolismo
12.
Nature ; 584(7821): 475-478, 2020 08.
Artigo em Inglês | MEDLINE | ID: mdl-32494008

RESUMO

The endoplasmic reticulum (ER) membrane complex (EMC) cooperates with the Sec61 translocon to co-translationally insert a transmembrane helix (TMH) of many multi-pass integral membrane proteins into the ER membrane, and it is also responsible for inserting the TMH of some tail-anchored proteins1-3. How EMC accomplishes this feat has been unclear. Here we report the first, to our knowledge, cryo-electron microscopy structure of the eukaryotic EMC. We found that the Saccharomyces cerevisiae EMC contains eight subunits (Emc1-6, Emc7 and Emc10), has a large lumenal region and a smaller cytosolic region, and has a transmembrane region formed by Emc4, Emc5 and Emc6 plus the transmembrane domains of Emc1 and Emc3. We identified a five-TMH fold centred around Emc3 that resembles the prokaryotic YidC insertase and that delineates a largely hydrophilic client protein pocket. The transmembrane domain of Emc4 tilts away from the main transmembrane region of EMC and is partially mobile. Mutational studies demonstrated that the flexibility of Emc4 and the hydrophilicity of the client pocket are required for EMC function. The EMC structure reveals notable evolutionary conservation with the prokaryotic insertases4,5, suggests that eukaryotic TMH insertion involves a similar mechanism, and provides a framework for detailed understanding of membrane insertion for numerous eukaryotic integral membrane proteins and tail-anchored proteins.


Assuntos
Microscopia Crioeletrônica , Retículo Endoplasmático/enzimologia , Membranas Intracelulares/enzimologia , Complexos Multiproteicos/química , Complexos Multiproteicos/ultraestrutura , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/ultraestrutura , Saccharomyces cerevisiae , Sítios de Ligação , Retículo Endoplasmático/química , Retículo Endoplasmático/ultraestrutura , Evolução Molecular , Interações Hidrofóbicas e Hidrofílicas , Membranas Intracelulares/química , Membranas Intracelulares/ultraestrutura , Modelos Moleculares , Complexos Multiproteicos/genética , Complexos Multiproteicos/metabolismo , Mutação , Domínios Proteicos , Subunidades Proteicas/química , Subunidades Proteicas/genética , Subunidades Proteicas/metabolismo , Saccharomyces cerevisiae/química , Saccharomyces cerevisiae/enzimologia , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/ultraestrutura , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Especificidade por Substrato
13.
Nature ; 584(7820): 304-309, 2020 08.
Artigo em Inglês | MEDLINE | ID: mdl-32581365

RESUMO

The human GABAB receptor-a member of the class C family of G-protein-coupled receptors (GPCRs)-mediates inhibitory neurotransmission and has been implicated in epilepsy, pain and addiction1. A unique GPCR that is known to require heterodimerization for function2-6, the GABAB receptor has two subunits, GABAB1 and GABAB2, that are structurally homologous but perform distinct and complementary functions. GABAB1 recognizes orthosteric ligands7,8, while GABAB2 couples with G proteins9-14. Each subunit is characterized by an extracellular Venus flytrap (VFT) module, a descending peptide linker, a seven-helix transmembrane domain and a cytoplasmic tail15. Although the VFT heterodimer structure has been resolved16, the structure of the full-length receptor and its transmembrane signalling mechanism remain unknown. Here we present a near full-length structure of the GABAB receptor, captured in an inactive state by cryo-electron microscopy. Our structure reveals several ligands that preassociate with the receptor, including two large endogenous phospholipids that are embedded within the transmembrane domains to maintain receptor integrity and modulate receptor function. We also identify a previously unknown heterodimer interface between transmembrane helices 3 and 5 of both subunits, which serves as a signature of the inactive conformation. A unique 'intersubunit latch' within this transmembrane interface maintains the inactive state, and its disruption leads to constitutive receptor activity.


Assuntos
Microscopia Crioeletrônica , Receptores de GABA-B/química , Receptores de GABA-B/ultraestrutura , Cálcio/metabolismo , Etanolaminas/química , Etanolaminas/metabolismo , Humanos , Ligantes , Modelos Moleculares , Fosforilcolina/química , Fosforilcolina/metabolismo , Domínios Proteicos , Multimerização Proteica , Subunidades Proteicas/química , Subunidades Proteicas/metabolismo , Receptores de GABA-B/metabolismo , Relação Estrutura-Atividade
14.
PLoS Genet ; 16(5): e1008681, 2020 05.
Artigo em Inglês | MEDLINE | ID: mdl-32463832

RESUMO

A large fraction of plant genomes is composed of transposable elements (TE), which provide a potential source of novel genes through "domestication"-the process whereby the proteins encoded by TE diverge in sequence, lose their ability to catalyse transposition and instead acquire novel functions for their hosts. In Arabidopsis, ANTAGONIST OF LIKE HETEROCHROMATIN PROTEIN 1 (ALP1) arose by domestication of the nuclease component of Harbinger class TE and acquired a new function as a component of POLYCOMB REPRESSIVE COMPLEX 2 (PRC2), a histone H3K27me3 methyltransferase involved in regulation of host genes and in some cases TE. It was not clear how ALP1 associated with PRC2, nor what the functional consequence was. Here, we identify ALP2 genetically as a suppressor of Polycomb-group (PcG) mutant phenotypes and show that it arose from the second, DNA binding component of Harbinger transposases. Molecular analysis of PcG compromised backgrounds reveals that ALP genes oppose silencing and H3K27me3 deposition at key PcG target genes. Proteomic analysis reveals that ALP1 and ALP2 are components of a variant PRC2 complex that contains the four core components but lacks plant-specific accessory components such as the H3K27me3 reader LIKE HETEROCHROMATION PROTEIN 1 (LHP1). We show that the N-terminus of ALP2 interacts directly with ALP1, whereas the C-terminus of ALP2 interacts with MULTICOPY SUPPRESSOR OF IRA1 (MSI1), a core component of PRC2. Proteomic analysis reveals that in alp2 mutant backgrounds ALP1 protein no longer associates with PRC2, consistent with a role for ALP2 in recruitment of ALP1. We suggest that the propensity of Harbinger TE to insert in gene-rich regions of the genome, together with the modular two component nature of their transposases, has predisposed them for domestication and incorporation into chromatin modifying complexes.


Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis , Proteínas do Grupo Polycomb/metabolismo , Proteínas Repressoras/metabolismo , Transposases/fisiologia , Animais , Arabidopsis/enzimologia , Arabidopsis/genética , Arabidopsis/metabolismo , Proteínas de Arabidopsis/química , Proteínas de Arabidopsis/genética , Domínio Catalítico/genética , Células Cultivadas , Domesticação , Regulação da Expressão Gênica de Plantas , Plantas Geneticamente Modificadas , Proteínas do Grupo Polycomb/genética , Ligação Proteica , Subunidades Proteicas/química , Subunidades Proteicas/genética , Subunidades Proteicas/metabolismo , Proteínas Repressoras/química , Proteínas Repressoras/genética , Células Sf9 , Spodoptera , Transposases/genética
15.
Nature ; 581(7809): 480-485, 2020 05.
Artigo em Inglês | MEDLINE | ID: mdl-32461643

RESUMO

Most proteins associate into multimeric complexes with specific architectures1,2, which often have functional properties such as cooperative ligand binding or allosteric regulation3. No detailed knowledge is available about how any multimer and its functions arose during evolution. Here we use ancestral protein reconstruction and biophysical assays to elucidate the origins of vertebrate haemoglobin, a heterotetramer of paralogous α- and ß-subunits that mediates respiratory oxygen transport and exchange by cooperatively binding oxygen with moderate affinity. We show that modern haemoglobin evolved from an ancient monomer and characterize the historical 'missing link' through which the modern tetramer evolved-a noncooperative homodimer with high oxygen affinity that existed before the gene duplication that generated distinct α- and ß-subunits. Reintroducing just two post-duplication historical substitutions into the ancestral protein is sufficient to cause strong tetramerization by creating favourable contacts with more ancient residues on the opposing subunit. These surface substitutions markedly reduce oxygen affinity and even confer cooperativity, because an ancient linkage between the oxygen binding site and the multimerization interface was already an intrinsic feature of the protein's structure. Our findings establish that evolution can produce new complex molecular structures and functions via simple genetic mechanisms that recruit existing biophysical features into higher-level architectures.


Assuntos
Evolução Molecular , Hemoglobinas/metabolismo , Regulação Alostérica , Sítios de Ligação/genética , Heme/metabolismo , Hemoglobinas/química , Humanos , Ferro/metabolismo , Modelos Moleculares , Oxigênio/metabolismo , Multimerização Proteica/genética , Estrutura Quaternária de Proteína/genética , Subunidades Proteicas/química , Subunidades Proteicas/metabolismo
16.
Biochim Biophys Acta Bioenerg ; 1861(8): 148215, 2020 08 01.
Artigo em Inglês | MEDLINE | ID: mdl-32360311

RESUMO

Marine Synechococcus are widespread in part because they are efficient at harvesting available light using their complex antenna, or phycobilisome, composed of multiple phycobiliproteins and bilin chromophores. Over 40% of Synechococcus strains are predicted to perform a type of chromatic acclimation that alters the ratio of two chromophores, green-light-absorbing phycoerythrobilin and blue-light-absorbing phycourobilin, to optimize light capture by phycoerythrin in the phycobilisome. Lyases are enzymes which catalyze the addition of bilin chromophores to specific cysteine residues on phycobiliproteins and are involved in chromatic acclimation. CpeY, a candidate lyase in the model strain Synechococcus sp. RS9916, added phycoerythrobilin to cysteine 82 of only the α subunit of phycoerythrin I (CpeA) in the presence or absence of the chaperone-like protein CpeZ in a recombinant protein expression system. These studies demonstrated that recombinant CpeY attaches phycoerythrobilin to as much as 72% of CpeA, making it one of the most efficient phycoerythrin lyases characterized to date. Phycobilisomes from a cpeY- mutant showed a near native bilin composition in all light conditions except for a slight replacement of phycoerythrobilin by phycourobilin at CpeA cysteine 82. This demonstrates that CpeY is not involved in any chromatic acclimation-driven chromophore changes and suggests that the chromophore attached at cysteine 82 of CpeA in the cpeY- mutant is ligated by an alternative phycoerythrobilin lyase. Although loss of CpeY does not greatly inhibit native phycobilisome assembly in vivo, the highly active recombinant CpeY can be used to generate large amounts of fluorescent CpeA for biotechnological uses.


Assuntos
Proteínas de Bactérias/metabolismo , Cisteína , Liases/metabolismo , Ficoeritrina/química , Subunidades Proteicas/química , Subunidades Proteicas/metabolismo , Synechococcus , Proteínas de Bactérias/genética , Liases/genética , Mutação
17.
Nat Commun ; 11(1): 2615, 2020 05 26.
Artigo em Inglês | MEDLINE | ID: mdl-32457314

RESUMO

F1Fo ATP synthase functions as a biological rotary generator that makes a major contribution to cellular energy production. It comprises two molecular motors coupled together by a central and a peripheral stalk. Proton flow through the Fo motor generates rotation of the central stalk, inducing conformational changes in the F1 motor that catalyzes ATP production. Here we present nine cryo-EM structures of E. coli ATP synthase to 3.1-3.4 Å resolution, in four discrete rotational sub-states, which provide a comprehensive structural model for this widely studied bacterial molecular machine. We observe torsional flexing of the entire complex and a rotational sub-step of Fo associated with long-range conformational changes that indicates how this flexibility accommodates the mismatch between the 3- and 10-fold symmetries of the F1 and Fo motors. We also identify density likely corresponding to lipid molecules that may contribute to the rotor/stator interaction within the Fo motor.


Assuntos
Proteínas de Escherichia coli/química , ATPases Translocadoras de Prótons/química , Difosfato de Adenosina/metabolismo , Microscopia Crioeletrônica , Proteínas de Escherichia coli/metabolismo , Lipídeos/química , Modelos Moleculares , Conformação Proteica , Subunidades Proteicas/química , Subunidades Proteicas/metabolismo , ATPases Translocadoras de Prótons/metabolismo , Rotação , Relação Estrutura-Atividade
18.
Nature ; 580(7802): 288-293, 2020 04.
Artigo em Inglês | MEDLINE | ID: mdl-32269335

RESUMO

Inactivation is the process by which ion channels terminate ion flux through their pores while the opening stimulus is still present1. In neurons, inactivation of both sodium and potassium channels is crucial for the generation of action potentials and regulation of firing frequency1,2. A cytoplasmic domain of either the channel or an accessory subunit is thought to plug the open pore to inactivate the channel via a 'ball-and-chain' mechanism3-7. Here we use cryo-electron microscopy to identify the molecular gating mechanism in calcium-activated potassium channels by obtaining structures of the MthK channel from Methanobacterium thermoautotrophicum-a purely calcium-gated and inactivating channel-in a lipid environment. In the absence of Ca2+, we obtained a single structure in a closed state, which was shown by atomistic simulations to be highly flexible in lipid bilayers at ambient temperature, with large rocking motions of the gating ring and bending of pore-lining helices. In Ca2+-bound conditions, we obtained several structures, including multiple open-inactivated conformations, further indication of a highly dynamic protein. These different channel conformations are distinguished by rocking of the gating rings with respect to the transmembrane region, indicating symmetry breakage across the channel. Furthermore, in all conformations displaying open channel pores, the N terminus of one subunit of the channel tetramer sticks into the pore and plugs it, with free energy simulations showing that this is a strong interaction. Deletion of this N terminus leads to functionally non-inactivating channels and structures of open states without a pore plug, indicating that this previously unresolved N-terminal peptide is responsible for a ball-and-chain inactivation mechanism.


Assuntos
Microscopia Crioeletrônica , Ativação do Canal Iônico , Methanobacterium/química , Canais de Potássio Cálcio-Ativados/antagonistas & inibidores , Canais de Potássio Cálcio-Ativados/ultraestrutura , Cálcio/metabolismo , Bicamadas Lipídicas/química , Bicamadas Lipídicas/metabolismo , Modelos Moleculares , Canais de Potássio Cálcio-Ativados/química , Canais de Potássio Cálcio-Ativados/metabolismo , Estrutura Secundária de Proteína , Subunidades Proteicas/química , Subunidades Proteicas/metabolismo , Termodinâmica
19.
Biochim Biophys Acta Gene Regul Mech ; 1863(7): 194560, 2020 07.
Artigo em Inglês | MEDLINE | ID: mdl-32302696

RESUMO

In mammalian cells, the SET1/MLL complexes are the main writers of the H3K4 methyl mark that is associated with active gene expression. The activities of these complexes are critically dependent on the association of the catalytic subunit with their shared core subunits, WDR5, RBBP5, ASH2L, and DPY30, collectively referred as WRAD. In addition, some of these core subunits can bind to proteins other than the SET1/MLL complex components. This review starts with discussion of the molecular activities of these core subunits, with an emphasis on DPY30 in organizing the assembly of the SET1/MLL complexes with other associated factors. This review then focuses on the roles of the core subunits in stem cells and development, as well as in diseased cell states, mainly cancer, and ends with discussion on dissecting the responsible activities of the core subunits and how we may target them for potential disease treatment. This article is part of a Special Issue entitled: The MLL family of proteins in normal development and disease edited by Thomas A Milne.


Assuntos
Regulação da Expressão Gênica no Desenvolvimento , Regulação Neoplásica da Expressão Gênica , Histona-Lisina N-Metiltransferase/metabolismo , Proteína de Leucina Linfoide-Mieloide/metabolismo , Multimerização Proteica , Animais , Histona-Lisina N-Metiltransferase/química , Humanos , Proteína de Leucina Linfoide-Mieloide/química , Subunidades Proteicas/química , Subunidades Proteicas/metabolismo
20.
Mol Cell Biol ; 40(13)2020 06 15.
Artigo em Inglês | MEDLINE | ID: mdl-32253346

RESUMO

Upstream activation factor (UAF) is a multifunctional transcription factor in Saccharomyces cerevisiae that plays dual roles in activating RNA polymerase I (Pol I) transcription and repression of Pol II. For Pol I, UAF binds to a specific upstream element in the ribosomal DNA (rDNA) promoter and interacts with two other Pol I initiation factors, the TATA-binding protein (TBP) and core factor (CF). We used an integrated combination of chemical cross-linking mass spectrometry (CXMS), molecular genetics, protein biochemistry, and structural modeling to understand the topological framework responsible for UAF complex formation. Here, we report the molecular topology of the UAF complex, describe new structural and functional domains that play roles in UAF complex integrity, assembly, and biological function, and provide roles for previously identified UAF domains that include the Rrn5 SANT and histone fold domains. We highlight the role of new domains in Uaf30 that include an N-terminal winged helix domain and a disordered tethering domain as well as a BORCS6-like domain found in Rrn9. Together, our results reveal a unique network of topological features that coalesce around a histone tetramer-like core to form the dual-function UAF complex.


Assuntos
Proteínas de Ligação a DNA/metabolismo , RNA Polimerase I/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Fatores de Transcrição/metabolismo , Reagentes para Ligações Cruzadas/química , Proteínas de Ligação a DNA/química , Espectrometria de Massas , Modelos Moleculares , Domínios Proteicos , Subunidades Proteicas/química , Subunidades Proteicas/metabolismo , Saccharomyces cerevisiae/química , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/química , Fatores de Transcrição/química , Ativação Transcricional
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