RESUMO
Short-range interactions and long-range contacts drive the 3D folding of structured proteins. The proteins' structure has a direct impact on their biological function. However, nearly 40% of the eukaryotes proteome is composed of intrinsically disordered proteins (IDPs) and protein regions that fluctuate between ensembles of numerous conformations. Therefore, to understand their biological function, it is critical to depict how the structural ensemble statistics correlate to the IDPs' amino acid sequence. Here, using small-angle X-ray scattering and time-resolved Förster resonance energy transfer (trFRET), we study the intramolecular structural heterogeneity of the neurofilament low intrinsically disordered tail domain (NFLt). Using theoretical results of polymer physics, we find that the Flory scaling exponent of NFLt subsegments correlates linearly with their net charge, ranging from statistics of ideal to self-avoiding chains. Surprisingly, measuring the same segments in the context of the whole NFLt protein, we find that regardless of the peptide sequence, the segments' structural statistics are more expanded than when measured independently. Our findings show that while polymer physics can, to some level, relate the IDP's sequence to its ensemble conformations, long-range contacts between distant amino acids play a crucial role in determining intramolecular structures. This emphasizes the necessity of advanced polymer theories to fully describe IDPs ensembles with the hope that it will allow us to model their biological function.
Assuntos
Proteínas Intrinsicamente Desordenadas , Proteínas Intrinsicamente Desordenadas/química , Conformação Proteica , Sequência de Aminoácidos , Eucariotos/metabolismo , PolímerosRESUMO
This study combines molecular dynamics (MD) simulations with small angle x-ray scattering (SAXS) measurements to investigate the range of conformations that can be adopted by a pH/ionic strength (IS) sensitive protein and to quantify its distinct populations in solution. To explore how the conformational distribution of proteins may be modified in the environmental niches of biological media, we focus on the periplasmic ferric binding protein A (FbpA) from Haemophilus influenzae involved in the mechanism by which bacteria capture iron from higher organisms. We examine iron-binding/release mechanisms of FbpA in varying conditions simulating its biological environment. While we show that these changes fall within the detectable range for SAXS as evidenced by differences observed in the theoretical scattering patterns calculated from the crystal structure models of apo and holo forms, detection of conformational changes due to the point mutation D52A and changes in ionic strength (IS) from SAXS scattering profiles have been challenging. Here, to reach conclusions, statistical analyses with SAXS profiles and results from different techniques were combined in a complementary fashion. The SAXS data complemented by size exclusion chromatography point to multiple and/or alternative conformations at physiological IS, whereas they are well-explained by single crystallographic structures in low IS buffers. By fitting the SAXS data with unique conformations sampled by a series of MD simulations under conditions mimicking the buffers, we quantify the populations of the occupied substates. We also find that the D52A mutant that we predicted by coarse-grained computational modeling to allosterically control the iron binding site in FbpA, responds to the environmental changes in our experiments with conformational selection scenarios that differ from those of the wild type.
Assuntos
Proteínas de Bactérias , Simulação de Dinâmica Molecular , Espalhamento a Baixo Ângulo , Raios X , Difração de Raios X , FerroRESUMO
As the decline of malaria cases stalled over the last five years, novel targets in Plasmodium falciparum are necessary for the development of new drugs. Glycogen Synthase Kinase (PfGSK3) has been identified as a potential target, since its selective inhibitors were shown to disrupt the parasitès life cycle. In the uncanonical N-terminal region of the parasite enzyme, we identified several autophosphorylation sites and probed their role in activity regulation of PfGSK3. By combining molecular modeling with experimental small-angle X-ray scattering data, we show that increased PfGSK3 activity is promoted by conformational changes in the PfGSK3 N-terminus, triggered by N-terminal phosphorylation. Our work provides novel insights into the structure and regulation of the malarial PfGSK3.
Assuntos
Quinase 3 da Glicogênio Sintase/metabolismo , Malária Falciparum/metabolismo , Modelos Moleculares , Plasmodium falciparum/enzimologia , Transdução de Sinais , Biologia Computacional/métodos , Ativação Enzimática , Escherichia coli/genética , Escherichia coli/metabolismo , Quinase 3 da Glicogênio Sintase/genética , Malária Falciparum/parasitologia , Fosforilação , Espalhamento a Baixo Ângulo , Difração de Raios X/métodosRESUMO
The binding of lipoprotein lipase (LPL) to GPIHBP1 focuses the intravascular hydrolysis of triglyceride-rich lipoproteins on the surface of capillary endothelial cells. This process provides essential lipid nutrients for vital tissues (e.g., heart, skeletal muscle, and adipose tissue). Deficiencies in either LPL or GPIHBP1 impair triglyceride hydrolysis, resulting in severe hypertriglyceridemia. The activity of LPL in tissues is regulated by angiopoietin-like proteins 3, 4, and 8 (ANGPTL). Dogma has held that these ANGPTLs inactivate LPL by converting LPL homodimers into monomers, rendering them highly susceptible to spontaneous unfolding and loss of enzymatic activity. Here, we show that binding of an LPL-specific monoclonal antibody (5D2) to the tryptophan-rich lipid-binding loop in the carboxyl terminus of LPL prevents homodimer formation and forces LPL into a monomeric state. Of note, 5D2-bound LPL monomers are as stable as LPL homodimers (i.e., they are not more prone to unfolding), but they remain highly susceptible to ANGPTL4-catalyzed unfolding and inactivation. Binding of GPIHBP1 to LPL alone or to 5D2-bound LPL counteracts ANGPTL4-mediated unfolding of LPL. In conclusion, ANGPTL4-mediated inactivation of LPL, accomplished by catalyzing the unfolding of LPL, does not require the conversion of LPL homodimers into monomers. Thus, our findings necessitate changes to long-standing dogma on mechanisms for LPL inactivation by ANGPTL proteins. At the same time, our findings align well with insights into LPL function from the recent crystal structure of the LPLâ¢GPIHBP1 complex.
Assuntos
Proteína 4 Semelhante a Angiopoietina/metabolismo , Lipase Lipoproteica/química , Triglicerídeos/sangue , Motivos de Aminoácidos , Proteína 4 Semelhante a Angiopoietina/genética , Animais , Anticorpos Monoclonais/metabolismo , Dimerização , Humanos , Hipertrigliceridemia/enzimologia , Hipertrigliceridemia/genética , Hipertrigliceridemia/metabolismo , Lipase Lipoproteica/genética , Lipase Lipoproteica/metabolismo , Desdobramento de Proteína , Receptores de Lipoproteínas/química , Receptores de Lipoproteínas/genética , Receptores de Lipoproteínas/metabolismoRESUMO
Lipoprotein lipase (LPL) is responsible for the intravascular processing of triglyceride-rich lipoproteins. The LPL within capillaries is bound to GPIHBP1, an endothelial cell protein with a three-fingered LU domain and an N-terminal intrinsically disordered acidic domain. Loss-of-function mutations in LPL or GPIHBP1 cause severe hypertriglyceridemia (chylomicronemia), but structures for LPL and GPIHBP1 have remained elusive. Inspired by our recent discovery that GPIHBP1's acidic domain preserves LPL structure and activity, we crystallized an LPL-GPIHBP1 complex and solved its structure. GPIHBP1's LU domain binds to LPL's C-terminal domain, largely by hydrophobic interactions. Analysis of electrostatic surfaces revealed that LPL contains a large basic patch spanning its N- and C-terminal domains. GPIHBP1's acidic domain was not defined in the electron density map but was positioned to interact with LPL's large basic patch, providing a likely explanation for how GPIHBP1 stabilizes LPL. The LPL-GPIHBP1 structure provides insights into mutations causing chylomicronemia.
Assuntos
Lipase Lipoproteica/metabolismo , Plasma/metabolismo , Receptores de Lipoproteínas/metabolismo , Triglicerídeos/sangue , Triglicerídeos/metabolismo , Animais , Células CHO , Capilares/metabolismo , Linhagem Celular , Cricetulus , Cristalografia por Raios X/métodos , Células Endoteliais/metabolismo , Humanos , Hidrólise , Hipertrigliceridemia/metabolismoRESUMO
The urokinase receptor (uPAR) is a founding member of a small protein family with multiple Ly6/uPAR (LU) domains. The motif defining these LU domains contains five plesiotypic disulfide bonds stabilizing its prototypical three-fingered fold having three protruding loops. Notwithstanding the detailed knowledge on structure-function relationships in uPAR, one puzzling enigma remains unexplored. Why does the first LU domain in uPAR (DI) lack one of its consensus disulfide bonds, when the absence of this particular disulfide bond impairs the correct folding of other single LU domain-containing proteins? Here, using a variety of contemporary biophysical methods, we found that reintroducing the two missing half-cystines in uPAR DI caused the spontaneous formation of the corresponding consensus 7-8 LU domain disulfide bond. Importantly, constraints due to this cross-link impaired (i) the binding of uPAR to its primary ligand urokinase and (ii) the flexible interdomain assembly of the three LU domains in uPAR. We conclude that the evolutionary deletion of this particular disulfide bond in uPAR DI may have enabled the assembly of a high-affinity urokinase-binding cavity involving all three LU domains in uPAR. Of note, an analogous neofunctionalization occurred in snake venom α-neurotoxins upon loss of another pair of the plesiotypic LU domain half-cystines. In summary, elimination of the 7-8 consensus disulfide bond in the first LU domain of uPAR did have significant functional and structural consequences.
Assuntos
Evolução Biológica , Deleção de Sequência , Sulfetos/metabolismo , Ativador de Plasminogênio Tipo Uroquinase/metabolismo , Sequência de Aminoácidos , Sítios de Ligação , Fenômenos Biofísicos , Quimotripsina/metabolismo , Glicosilação , Cinética , Ligantes , Dobramento de Proteína , Proteólise , Homologia de Sequência de Aminoácidos , Relação Estrutura-Atividade , Ativador de Plasminogênio Tipo Uroquinase/químicaRESUMO
Plant growth and survival depend upon the activity of membrane transporters that control the movement and distribution of solutes into, around, and out of plants. Although many plant transporters are known, their intrinsic properties make them difficult to study. In barley (Hordeum vulgare), the root anion-permeable transporter Bot1 plays a key role in tolerance to high soil boron, facilitating the efflux of borate from cells. However, its three-dimensional structure is unavailable and the molecular basis of its permeation function is unknown. Using an integrative platform of computational, biophysical, and biochemical tools as well as molecular biology, electrophysiology, and bioinformatics, we provide insight into the origin of transport function of Bot1. An atomistic model, supported by atomic force microscopy measurements, reveals that the protein folds into 13 transmembrane-spanning and five cytoplasmic α-helices. We predict a trimeric assembly of Bot1 and the presence of a Na(+) ion binding site, located in the proximity of a pore that conducts anions. Patch-clamp electrophysiology of Bot1 detects Na(+)-dependent polyvalent anion transport in a Nernstian manner with channel-like characteristics. Using alanine scanning, molecular dynamics simulations, and transport measurements, we show that conductance by Bot1 is abolished by removal of the Na(+) ion binding site. Our data enhance the understanding of the permeation functions of Bot1.
Assuntos
Hordeum/metabolismo , Proteínas de Membrana Transportadoras/metabolismo , Proteínas de Plantas/metabolismo , Sódio/metabolismo , Ânions/metabolismo , Sítios de Ligação , Boratos/metabolismo , Sistema Livre de Células , Simulação por Computador , Bicamadas Lipídicas/metabolismo , Lipossomos/metabolismo , Proteínas de Membrana Transportadoras/química , Modelos Moleculares , Permeabilidade , Pichia/metabolismo , Proteínas de Plantas/química , Dobramento de Proteína , Multimerização Proteica , Triticum/metabolismoRESUMO
Glycyl tRNA synthetase (GlyRS) provides a unique case among class II aminoacyl tRNA synthetases, with two clearly widespread types of enzymes: a dimeric (α2) species present in some bacteria, archaea, and eukaryotes; and a heterotetrameric form (α2ß2) present in most bacteria. Although the differences between both types of GlyRS at the anticodon binding domain level are evident, the extent and implications of the variations in the catalytic domain have not been described, and it is unclear whether the mechanism of amino acid recognition is also dissimilar. Here, we show that the α-subunit of the α2ß2 GlyRS from the bacterium Aquifex aeolicus is able to perform the first step of the aminoacylation reaction, which involves the activation of the amino acid with ATP. The crystal structure of the α-subunit in the complex with an analog of glycyl adenylate at 2.8 Å resolution presents a conformational arrangement that properly positions the cognate amino acid. This work shows that glycine is recognized by a subset of different residues in the two types of GlyRS. A structural and sequence analysis of class II catalytic domains shows that bacterial GlyRS is closely related to alanyl tRNA synthetase, which led us to define a new subclassification of these ancient enzymes and to propose an evolutionary path of α2ß2 GlyRS, convergent with α2 GlyRS and divergent from AlaRS, thus providing a possible explanation for the puzzling existence of two proteins sharing the same fold and function but not a common ancestor.
Assuntos
Glicina-tRNA Ligase/química , Filogenia , Bactérias/enzimologia , Cristalografia por Raios X , Modelos Moleculares , Conformação ProteicaRESUMO
Bacteriophages produce endolysins, which lyse the bacterial host cell to release newly produced virions. The timing of lysis is regulated and is thought to involve the activation of a molecular switch. We present a crystal structure of the activated endolysin CTP1L that targets Clostridium tyrobutyricum, consisting of a complex between the full-length protein and an N-terminally truncated C-terminal cell wall binding domain (CBD). The truncated CBD is produced through an internal translation start site within the endolysin gene. Mutants affecting the internal translation site change the oligomeric state of the endolysin and reduce lytic activity. The activity can be modulated by reconstitution of the full-length endolysin-CBD complex with free CBD. The same oligomerization mechanism applies to the CD27L endolysin that targets Clostridium difficile and the CS74L endolysin that targets Clostridium sporogenes. When the CTP1L endolysin gene is introduced into the commensal bacterium Lactococcus lactis, the truncated CBD is also produced, showing that the alternative start codon can be used in other bacterial species. The identification of a translational switch affecting oligomerization presented here has implications for the design of effective endolysins for the treatment of bacterial infections.
Assuntos
Endopeptidases/química , Sequência de Aminoácidos , Bacteriófagos/enzimologia , Bacteriófagos/genética , Clostridium tyrobutyricum/efeitos dos fármacos , Códon de Iniciação , Endopeptidases/genética , Endopeptidases/metabolismo , Endopeptidases/toxicidade , Dados de Sequência Molecular , Mutação , Ligação Proteica , Multimerização ProteicaRESUMO
Small-angle X-ray scattering (SAXS) and Nuclear Magnetic Resonance (NMR) are established methods to analyze the structure and structural transitions of biological macromolecules in solution. Both methods are directly applicable to near-native macromolecular solutions and allow one to study structural responses to physical and chemical changes or ligand additions. Whereas SAXS is applied to elucidate overall structure, interactions and flexibility over a wide range of particle sizes, NMR yields atomic resolution detail for moderately sized macromolecules. NMR is arguably the most powerful technique for the experimental analysis of dynamics. The joint application of these two highly complementary techniques provides an extremely useful approach that facilitates comprehensive characterization of biomacromolecular solutions.
Assuntos
Substâncias Macromoleculares/química , Substâncias Macromoleculares/metabolismo , Ressonância Magnética Nuclear Biomolecular/métodos , Espalhamento a Baixo Ângulo , Difração de Raios X/métodos , Estatística como AssuntoRESUMO
The extracellular accumulation of amyloid ß (Aß) peptides is characteristic of Alzheimer's disease (AD). However, formation of diffusible, oligomeric forms of Aß, both on and off pathways to amyloid fibrils, is thought to include neurotoxic species responsible for synaptic loss and neurodegeneration, rather than polymeric amyloid aggregates. The 8-hydroxyquinolines (8-HQ) clioquinol (CQ) and PBT2 were developed for their ability to inhibit metal-mediated generation of reactive oxygen species from Aß:Cu complexes and have both undergone preclinical and Phase II clinical development for the treatment of AD. Their respective modes of action are not fully understood and may include both inhibition of Aß fibrillar polymerization and direct depolymerization of existing Aß fibrils. In the present study, we find that CQ and PBT2 can interact directly with Aß and affect its propensity to aggregate. Using a combination of biophysical techniques, we demonstrate that, in the presence of these 8-HQs and in the absence of metal ions, Aß associates with two 8-HQ molecules and forms a dimer. Furthermore, 8-HQ bind Aß with an affinity of 1-10 µm and suppress the formation of large (>30 kDa) oligomers. The stabilized low molecular weight species are nontoxic. Treatment with 8-HQs also reduces the levels of in vivo soluble oligomers in a Caenorhabditis elegans model of Aß toxicity. We propose that 8-HQs possess an additional mechanism of action that neutralizes neurotoxic Aß oligomer formation through stabilization of small (dimeric) nontoxic Aß conformers.
Assuntos
Doença de Alzheimer/metabolismo , Peptídeos beta-Amiloides/metabolismo , Hidroxiquinolinas/metabolismo , Fragmentos de Peptídeos/metabolismo , Peptídeos beta-Amiloides/química , Peptídeos beta-Amiloides/ultraestrutura , Animais , Benzotiazóis , Biofísica , Caenorhabditis elegans , Células Cultivadas , Córtex Cerebral/citologia , Cromatografia em Gel , Clioquinol/análogos & derivados , Clioquinol/metabolismo , Ensaio de Imunoadsorção Enzimática , Humanos , Camundongos , Microscopia Eletrônica , Neurônios/efeitos dos fármacos , Neurônios/metabolismo , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/ultraestrutura , Ligação Proteica/efeitos dos fármacos , Espalhamento a Baixo Ângulo , Tiazóis/metabolismoRESUMO
Ethylene initiates important aspects of plant growth and development through disulfide-linked receptor dimers located in the endoplasmic reticulum. The receptors feature a small transmembrane, ethylene binding domain followed by a large cytosolic domain, which serves as a scaffold for the assembly of large molecular weight complexes of different ethylene receptors and other cellular participants of the ethylene signaling pathway. Here we report the crystallographic structures of the ethylene receptor 1 (ETR1) catalytic ATP-binding and the ethylene response sensor 1 dimerization histidine phosphotransfer (DHp) domains and the solution structure of the entire cytosolic domain of ETR1, all from Arabidopsis thaliana. The isolated dimeric ethylene response sensor 1 DHp domain is asymmetric, the result of different helical bending angles close to the conserved His residue. The structures of the catalytic ATP-binding, DHp, and receiver domains of ethylene receptors and of a homologous, but dissimilar, GAF domain were refined against experimental small angle x-ray scattering data, leading to a structural model of the entire cytosolic domain of the ethylene receptor 1. The model illustrates that the cytosolic domain is shaped like a dumbbell and that the receiver domain is flexible and assumes a position different from those observed in prokaryotic histidine kinases. Furthermore the cytosolic domain of ETR1 plays a key role, interacting with all other receptors and several participants of the ethylene signaling pathway. Our model, therefore, provides the first step toward a detailed understanding of the molecular mechanics of this important signal transduction process in plants.
Assuntos
Proteínas de Plantas/química , Receptores de Superfície Celular/química , Arabidopsis/metabolismo , Cristalografia por Raios X , Citosol/metabolismo , Etilenos/metabolismo , Regulação da Expressão Gênica de Plantas , Proteínas de Plantas/metabolismo , Estrutura Secundária de Proteína , Receptores de Superfície Celular/metabolismoRESUMO
The bacteriophage ΦCD27 is capable of lysing Clostridium difficile, a pathogenic bacterium that is a major cause for nosocomial infection. A recombinant CD27L endolysin lyses C. difficile in vitro, and represents a promising alternative as a bactericide. To better understand the lysis mechanism, we have determined the crystal structure of an autoproteolytic fragment of the CD27L endolysin. The structure covers the C-terminal domain of the endolysin, and represents a novel fold that is identified in a number of lysins that target Clostridia bacteria. The structure indicates endolysin cleavage occurs at the stem of the linker connecting the catalytic domain with the C-terminal domain. We also solved the crystal structure of the C-terminal domain of a slow cleaving mutant of the CTP1L endolysin that targets C. tyrobutyricum. Two distinct dimerization modes are observed in the crystal structures for both endolysins, despite a sequence identity of only 22% between the domains. The dimers are validated to be present for the full length protein in solution by right angle light scattering, small angle X-ray scattering and cross-linking experiments using the cross-linking amino acid p-benzoyl-L-phenylalanine (pBpa). Mutagenesis on residues contributing to the dimer interfaces indicates that there is a link between the dimerization modes and the autocleavage mechanism. We show that for the CTP1L endolysin, there is a reduction in lysis efficiency that is proportional to the cleavage efficiency. We propose a model for endolysin triggering, where the extended dimer presents the inactive state, and a switch to the side-by-side dimer triggers the cleavage of the C-terminal domain. This leads to the release of the catalytic portion of the endolysin, enabling the efficient digestion of the bacterial cell wall.
Assuntos
Bacteriófagos , Clostridioides difficile , Endopeptidases , Modelos Biológicos , Proteínas Virais , Bacteriófagos/enzimologia , Bacteriófagos/genética , Clostridioides difficile/química , Clostridioides difficile/genética , Clostridioides difficile/metabolismo , Clostridioides difficile/virologia , Cristalografia por Raios X , Endopeptidases/química , Endopeptidases/genética , Endopeptidases/metabolismo , Humanos , Estrutura Terciária de Proteína , Proteínas Virais/química , Proteínas Virais/genética , Proteínas Virais/metabolismoRESUMO
To survive and replicate within the human host, malaria parasites must invade erythrocytes. Invasion can be mediated by the P. falciparum reticulocyte-binding homologue protein 4 (PfRh4) on the merozoite surface interacting with complement receptor type 1 (CR1, CD35) on the erythrocyte membrane. The PfRh4 attachment site lies within the three N-terminal complement control protein modules (CCPs 1-3) of CR1, which intriguingly also accommodate binding and regulatory sites for the key complement activation-specific proteolytic products, C3b and C4b. One of these regulatory activities is decay-accelerating activity. Although PfRh4 does not impact C3b/C4b binding, it does inhibit this convertase disassociating capability. Here, we have employed ELISA, co-immunoprecipitation, and surface plasmon resonance to demonstrate that CCP 1 contains all the critical residues for PfRh4 interaction. We fine mapped by homologous substitution mutagenesis the PfRh4-binding site on CCP 1 and visualized it with a solution structure of CCPs 1-3 derived by NMR and small angle x-ray scattering. We cross-validated these results by creating an artificial PfRh4-binding site through substitution of putative PfRh4-interacting residues from CCP 1 into their homologous positions within CCP 8; strikingly, this engineered binding site had an â¼30-fold higher affinity for PfRh4 than the native one in CCP 1. These experiments define a candidate site on CR1 by which P. falciparum merozoites gain access to human erythrocytes in a non-sialic acid-dependent pathway of merozoite invasion.
Assuntos
Proteínas de Membrana/metabolismo , Merozoítos/metabolismo , Plasmodium falciparum/metabolismo , Proteínas de Protozoários/metabolismo , Receptores de Complemento 3b/metabolismo , Sítios de Ligação , Complemento C3b/química , Complemento C3b/genética , Complemento C3b/metabolismo , Complemento C4b/química , Complemento C4b/genética , Complemento C4b/metabolismo , Eritrócitos/química , Eritrócitos/metabolismo , Eritrócitos/parasitologia , Células HEK293 , Humanos , Proteínas de Membrana/química , Proteínas de Membrana/genética , Merozoítos/química , Mutagênese , Ressonância Magnética Nuclear Biomolecular , Plasmodium falciparum/química , Plasmodium falciparum/genética , Proteínas de Protozoários/química , Proteínas de Protozoários/genética , Receptores de Complemento 3b/química , Receptores de Complemento 3b/genética , Espalhamento a Baixo Ângulo , Ressonância de Plasmônio de Superfície , Difração de Raios XRESUMO
Ribulose-bisphosphate carboxylase/oxygenase (Rubisco) activase uses the energy from ATP hydrolysis to remove tight binding inhibitors from Rubisco, thus playing a key role in regulating photosynthesis in plants. Although several structures have recently added much needed structural information for different Rubisco activase enzymes, the arrangement of these subunits in solution remains unclear. In this study, we use a variety of techniques to show that Rubisco activase forms a wide range of structures in solution, ranging from monomers to much higher order species, and that the distribution of these species is highly dependent on protein concentration. The data support a model in which Rubisco activase forms an open spiraling structure rather than a closed hexameric structure. At protein concentrations of 1 µM, corresponding to the maximal activity of the enzyme, Rubisco activase has an oligomeric state of 2-4 subunits. We propose a model in which Rubisco activase requires at least 1 neighboring subunit for hydrolysis of ATP.
Assuntos
Proteínas de Plantas/química , Proteínas de Plantas/metabolismo , Multimerização Proteica , Estrutura Quaternária de Proteína , Trifosfato de Adenosina/metabolismo , Ativação Enzimática , Hidrólise , Modelos Moleculares , Proteínas de Plantas/genética , Subunidades Proteicas/química , Subunidades Proteicas/genética , Subunidades Proteicas/metabolismo , Espalhamento a Baixo Ângulo , Soluções/química , Nicotiana/enzimologia , Nicotiana/genética , Difração de Raios XRESUMO
A membrane-embedded curdlan synthase (CrdS) from Agrobacterium is believed to catalyse a repetitive addition of glucosyl residues from UDP-glucose to produce the (1,3)-ß-d-glucan (curdlan) polymer. We report wheat germ cell-free protein synthesis (WG-CFPS) of full-length CrdS containing a 6xHis affinity tag and either Factor Xa or Tobacco Etch Virus proteolytic sites, using a variety of hydrophobic membrane-mimicking environments. Full-length CrdS was synthesised with no variations in primary structure, following analysis of tryptic fragments by MALDI-TOF/TOF Mass Spectrometry. Preparative scale WG-CFPS in dialysis mode with Brij-58 yielded CrdS in mg/ml quantities. Analysis of structural and functional properties of CrdS during protein synthesis showed that CrdS was co-translationally inserted in DMPC liposomes during WG-CFPS, and these liposomes could be purified in a single step by density gradient floatation. Incorporated CrdS exhibited a random orientation topology. Following affinity purification of CrdS, the protein was reconstituted in nanodiscs with Escherichia coli lipids or POPC and a membrane scaffold protein MSP1E3D1. CrdS nanodiscs were characterised by small-angle X-ray scattering using synchrotron radiation and the data obtained were consistent with insertion of CrdS into bilayers. We found CrdS synthesised in the presence of the Ac-AAAAAAD surfactant peptide or co-translationally inserted in liposomes made from E. coli lipids to be catalytically competent. Conversely, CrdS synthesised with only Brij-58 was inactive. Our findings pave the way for future structural studies of this industrially important catalytic membrane protein.
Assuntos
Glucosiltransferases/química , Lipossomos/química , Nanopartículas/química , Nanotecnologia/métodos , beta-Glucanas/química , Agrobacterium/metabolismo , Catálise , Sistema Livre de Células , Escherichia coli/metabolismo , Glucose/química , Microscopia Eletrônica de Transmissão/métodos , Peptídeos/química , Plasmídeos/metabolismo , Biossíntese de Proteínas , Proteínas/química , RNA Mensageiro/metabolismo , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Tensoativos/química , Tripsina/química , Difosfato de Uridina/químicaRESUMO
The major myelin protein expressed by the peripheral nervous system Schwann cells is protein zero (P0), which represents 50% of the total protein content in myelin. This 30-kDa integral membrane protein consists of an immunoglobulin (Ig)-like domain, a transmembrane helix, and a 69-residue C-terminal cytoplasmic tail (P0ct). The basic residues in P0ct contribute to the tight packing of myelin lipid bilayers, and alterations in the tail affect how P0 functions as an adhesion molecule necessary for the stability of compact myelin. Several neurodegenerative neuropathies are related to P0, including the more common Charcot-Marie-Tooth disease (CMT) and Dejerine-Sottas syndrome (DSS) as well as rare cases of motor and sensory polyneuropathy. We found that high P0ct concentrations affected the membrane properties of bicelles and induced a lamellar-to-inverted hexagonal phase transition, which caused bicelles to fuse into long, protein-containing filament-like structures. These structures likely reflect the formation of semicrystalline lipid domains with potential relevance for myelination. Not only is P0ct important for stacking lipid membranes, but time-lapse fluorescence microscopy also shows that it might affect membrane properties during myelination. We further describe recombinant production and low-resolution structural characterization of full-length human P0. Our findings shed light on P0ct effects on membrane properties, and with the successful purification of full-length P0, we have new tools to study the role of P0 in myelin formation and maintenance in vitro.
Assuntos
Proteína P0 da Mielina , Proteína P0 da Mielina/metabolismo , Proteína P0 da Mielina/química , Proteína P0 da Mielina/genética , Humanos , Bainha de Mielina/metabolismo , Bainha de Mielina/química , Bicamadas Lipídicas/química , Bicamadas Lipídicas/metabolismo , Lipídeos de Membrana/metabolismo , Lipídeos de Membrana/química , Citoplasma/metabolismo , Citoplasma/químicaRESUMO
While the elucidation of regulatory mechanisms of folded proteins is facilitated due to their amenability to high-resolution structural characterization, investigation of these mechanisms in disordered proteins is more challenging due to their structural heterogeneity, which can be captured by a variety of biophysical approaches. Here, we used the transcriptional master corepressor CtBP, which binds the putative metastasis suppressor RAI2 through repetitive SLiMs, as a model system. Using cryo-electron microscopy embedded in an integrative structural biology approach, we show that RAI2 unexpectedly induces CtBP polymerization through filaments of stacked tetrameric CtBP layers. These filaments lead to RAI2-mediated CtBP nuclear foci and relieve its corepressor function in RAI2-expressing cancer cells. The impact of RAI2-mediated CtBP loss-of-function is illustrated by the analysis of a diverse cohort of prostate cancer patients, which reveals a substantial decrease in RAI2 in advanced treatment-resistant cancer subtypes. As RAI2-like SLiM motifs are found in a wide range of organisms, including pathogenic viruses, our findings serve as a paradigm for diverse functional effects through multivalent interaction-mediated polymerization by disordered proteins in healthy and diseased conditions.
Assuntos
Oxirredutases do Álcool , Polimerização , Neoplasias da Próstata , Humanos , Masculino , Neoplasias da Próstata/metabolismo , Neoplasias da Próstata/genética , Neoplasias da Próstata/patologia , Oxirredutases do Álcool/metabolismo , Oxirredutases do Álcool/genética , Oxirredutases do Álcool/química , Microscopia Crioeletrônica , Linhagem Celular Tumoral , Proteínas de Ligação a DNA/metabolismo , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/química , Ligação Proteica , Células HEK293 , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas Adaptadoras de Transdução de Sinal/química , Motivos de Aminoácidos , Proteínas Correpressoras/metabolismo , Proteínas Correpressoras/genéticaRESUMO
The urokinase-type plasminogen activator receptor (uPAR) provides a rendezvous between proteolytic degradation of the extracellular matrix and integrin-mediated adhesion to vitronectin. These processes are, however, tightly linked because the high affinity binding of urokinase regulates the binding of uPAR to matrix-embedded vitronectin. Although crystal structures exist to define the corresponding static bi- and trimolecular receptor complexes, it is evident that the dynamic property of uPAR plays a decisive role in its function. In the present study, we combine small angle x-ray scattering, hydrogen-deuterium exchange, and surface plasmon resonance to develop a structural model describing the allosteric regulation of uPAR. We show that the flexibility of its N-terminal domain provides the key for understanding this allosteric mechanism. Importantly, our model has direct implications for understanding uPAR-assisted cell adhesion and migration as well as for translational research, including targeted intervention therapy and non-invasive tumor imaging in vivo.
Assuntos
Matriz Extracelular , Proteólise , Receptores de Ativador de Plasminogênio Tipo Uroquinase , Vitronectina , Regulação Alostérica , Animais , Adesão Celular , Linhagem Celular , Movimento Celular , Medição da Troca de Deutério , Drosophila melanogaster , Matriz Extracelular/química , Matriz Extracelular/metabolismo , Humanos , Invasividade Neoplásica , Neoplasias/tratamento farmacológico , Neoplasias/metabolismo , Neoplasias/patologia , Ligação Proteica , Estrutura Terciária de Proteína , Receptores de Ativador de Plasminogênio Tipo Uroquinase/química , Receptores de Ativador de Plasminogênio Tipo Uroquinase/metabolismo , Espalhamento de Radiação , Relação Estrutura-Atividade , Vitronectina/química , Vitronectina/metabolismo , Raios XRESUMO
The ATSAS software suite provides a comprehensive set of programs for the processing, analysis and modeling of small-angle scattering data, tailored for but not limited to data acquired on biological macromolecules. In this review the major components and developments in the ATSAS package are described, with a focus on user driven application. Data reduction, analysis and modeling approaches and strategies will be introduced and discussed. At the time of writing the latest package, ATSAS 3.1, is freely available for academic users at: https://www.embl-hamburg.de/biosaxs/software.html.