381 results on '"Valpuesta, José M."'
Search Results
152. Biochemical Characterization of Symmetric GroEL-GroES Complexes
- Author
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Llorca, Oscar, primary, Carrascosa, José L., additional, and Valpuesta, José M., additional
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- 1996
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153. The formation of symmetrical GroEL-GroES complexes in the presence of ATP
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Llorca, Oscar, primary, Marco, Sergio, additional, Carrascosa, José L., additional, and Valpuesta, José M., additional
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- 1994
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154. DNA conformational change induced by the bacteriophage Φ29 connector
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Valpuesta, José M., primary, Serrano, Manuel, additional, Donate, Luis E., additional, Herranz, Lucía, additional, and Carrascosa, José L., additional
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- 1992
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155. Mechanical stability of low-humidity single DNA molecules.
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Hormeño, Silvia, Ibarra, Borja, Valpuesta, José M., Carrascosa, José L., and Ricardo Arias-Gonzalez, J.
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DNA electrostatic character is mostly determined by both water and counterions activities in the phosphate backbone, which together with base sequence, further confer its higher order structure. The authors overstretch individual double-stranded DNA molecules in water-ethanol solutions to investigate the modulation of its mechanical stability by hydration and polycations. The authors found that DNA denatures as ethanol concentration is increased and spermine concentration decreased. This is manifested by an increase in melting hysteresis between the stretch and release curves, with sharp transition at 10% ethanol and reentrant behavior at 60%, by a loss of cooperativity in the overstretching transition and by a dramatic decrease of both the persistence length and the flexural rigidity. Changes in base-stacking stability which are characteristic of the B-A transition between 70 and 80% ethanol concentration do not manifest in the mechanical properties of the double-helical molecule at low or high force or in the behavior of the overstretching and melting transitions within this ethanol concentration range. This is consistent with a mechanism in which A-type base-stacking is unstable in the presence of tension. Binding of motor proteins to DNA locally reduces the number of water molecules and therefore, our results may shed light on analogous reduced-water activity of DNA conditions caused by other molecules, which interact with DNA in vivo. © 2011 Wiley Periodicals, Inc. Biopolymers 97: 199-208, 2012. [ABSTRACT FROM AUTHOR]
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- 2012
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156. Crystal structure of the open conformation of the mammalian chaperonin CCT in complex with tubulin.
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Muñoz, Inés G, Yébenes, Hugo, Zhou, Min, Mesa, Pablo, Serna, Marina, Park, Ah Young, Bragado-Nilsson, Elisabeth, Beloso, Ana, de Cárcer, Guillermo, Malumbres, Marcos, Robinson, Carol V, Valpuesta, José M, and Montoya, Guillermo
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MOLECULAR chaperones ,CRYSTALS ,OLIGOMERS ,TUBULINS ,ELECTRON distribution ,ACTIN - Abstract
Protein folding is assisted by molecular chaperones. CCT (chaperonin containing TCP-1, or TRiC) is a 1-MDa oligomer that is built by two rings comprising eight different 60-kDa subunits. This chaperonin regulates the folding of important proteins including actin, α-tubulin and β-tubulin. We used an electron density map at 5.5 Å resolution to reconstruct CCT, which showed a substrate in the inner cavities of both rings. Here we present the crystal structure of the open conformation of this nanomachine in complex with tubulin, providing information about the mechanism by which it aids tubulin folding. The structure showed that the substrate interacts with loops in the apical and equatorial domains of CCT. The organization of the ATP-binding pockets suggests that the substrate is stretched inside the cavity. Our data provide the basis for understanding the function of this chaperonin. [ABSTRACT FROM AUTHOR]
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- 2011
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157. The structure of CCT–Hsc70NBD suggests a mechanism for Hsp70 delivery of substrates to the chaperonin.
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Cuéllar, Jorge, Martín-Benito, Jaime, Scheres, Sjors H. W., Sousa, Rui, Moro, Fernando, López-Viñas, Eduardo, Gómez-Puertas, Paulino, Muga, Arturo, Carrascosa, José L., and Valpuesta, José M.
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MOLECULAR biology ,HEAT shock proteins ,PROTEINS ,MOLECULAR chaperones ,NUCLEOTIDES ,PROKARYOTES - Abstract
Chaperones, a group of proteins that assist the folding of other proteins, seem to work in a coordinated manner. Two major chaperone families are heat-shock protein families Hsp60 and Hsp70. Here we show for the first time the formation of a stable complex between chaperonin-containing TCP-1 (CCT) and Hsc70, two eukaryotic representatives of these chaperone families. This interaction takes place between the apical domain of the CCTβ subunit and the nucleotide binding domain of Hsc70, and may serve to deliver the unfolded substrate from Hsc70 to the substrate binding region of CCT. We also show that a similar interaction does not occur between their prokaryotic counterparts GroEL and DnaK, suggesting that in eukarya the two types of chaperones have evolved to a concerted action that makes the folding task more efficient. [ABSTRACT FROM AUTHOR]
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- 2008
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158. Three-Dimensional Structure of Cloned T3 Connector Protein at 1.6nm Resolution
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Carrascosa, José L., primary, Valpuesta, José M., additional, and Fujisawa, Hisao, additional
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- 1990
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159. Structure of eukaryotic prefoldin and of its complexes with unfolded actin and the cytosolic chaperonin CCT.
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Martín-Benito, Jaime, Boskovic, Jasminka, Gómez-Puertas, Paulino, Carrascosa, José L., Simons, C.Torrey, Lewis, Sally A., Bartolini, Francesca, Cowan, Nicholas J., and Valpuesta, José M.
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EUKARYOTIC cells ,MOLECULAR chaperones ,ACTIN ,OLIGOMERS ,PROTEINS ,ELECTRON microscopy - Abstract
The biogenesis of the cytoskeletal proteins actin and tubulin involves interaction of nascent chains of each of the two proteins with the oligomeric protein prefoldin (PFD) and their subsequent transfer to the cytosolic chaperonin CCT (chaperonin containing TCP-1). Here we show by electron microscopy that eukaryotic PFD, which has a similar structure to its archaeal counterpart, interacts with unfolded actin along the tips of its projecting arms. In its PFD-bound state, actin seems to acquire a conformation similar to that adopted when it is bound to CCT. Three-dimensional reconstruction of the CCT:PFD complex based on cryoelectron microscopy reveals that PFD binds to each of the CCT rings in a unique conformation through two specific CCT subunits that are placed in a 1,4 arrangement. This defines the phasing of the CCT rings and suggests a handoff mechanism for PFD. [ABSTRACT FROM AUTHOR]
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- 2002
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160. The 'sequential allosteric ring' mechanism in the eukaryotic chaperonin-assisted folding of actin and tubulin.
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Llorca, Oscar, Martín-Benito, Jaime, Grantham, Julie, Ritco-Vonsovici, Monica, Willison, Keith R., Carrascosa, José L., and Valpuesta, José M.
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MOLECULAR chaperones ,TUBULINS ,ALLOSTERIC proteins ,PROTEINS ,ACTOMYOSIN ,CYTOSOL - Abstract
Folding to completion of actin and tubulin in the eukaryotic cytosol requires their interaction with cytosolic chaperonin CCT [containing tailless complex polypeptide 1 (TCP-1). Three-dimensional reconstructions of nucleotide-free CCT complexed to either actin or tubulin show that CCT stabilizes both cytoskeletal proteins in open and quasi-folded con-formations mediated through interactions that are both subunit specific and geometry dependent. Here we find that upon ATP binding, mimicked by the non-hydrolysable analog AMP-PNP (5'-adenylylimido-diphosphate), to both CCT-α-actin and CCT- β-tubulin complexes, the chaperonin component undergoes concerted movements of the apical domains, resulting in the cavity being closed off by the helical protrusions of the eight apical domains. However, in contrast to the GroE system, generation of this closed state does not induce the release of the substrate into the chaperonin cavity, and both cytoskeletal proteins remain bound to the chaperonin apical domains. Docking of the AMP-PNP-CCT-bound con-formations of a-actin and β-tubulin to their respective native atomic structures suggests that both proteins have progressed towards their native states. [ABSTRACT FROM AUTHOR]
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- 2001
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161. Eukaryotic chaperonin CCT stabilizes actin and tubulin folding intermediates in open quasi-native conformations.
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Llorca, Oscar, Martín-Benito, Jaime, Ritco-Vonsovici, Monica, Grantham, Julie, Hynes, Gillian M., Willison, Keith R., Carrascosa, José L., and Valpuesta, José M.
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ACTIN ,TUBULINS ,MOLECULAR chaperones ,ELECTRON microscopy ,PROTEIN folding ,ENZYME kinetics ,PROTEOMICS ,CYTOCHEMISTRY - Abstract
Three-dimensional reconstruction from cryoelectron micrographs of the eukaryotic cytosolic chaperonin CCT complexed to tubulin shows that CCT interacts with tubulin (both the α and β isoforms) using five specific CCT subunits. The CCT-tubulin interaction has a different geometry to the CCT-actin interaction, and a mixture of shared and unique CCT subunits is used in binding the two substrates. Docking of the atomic structures of both actin and tubulin to their CCT-bound conformation suggests a common mode of chaperonin-substrate interaction. CCT stabilizes quasi-native structures in both proteins that are open through their domain-connecting hinge regions, suggesting a novel mechanism and function of CCT in assisted protein folding. [ABSTRACT FROM AUTHOR]
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- 2000
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162. Synthesis of Mesoporous Silica Coated Gold Nanorods Loaded with Methylene Blue and Its Potentials in Antibacterial Applications.
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Fernández-Lodeiro, Adrián, Djafari, Jamila, Fernández-Lodeiro, Javier, Duarte, Maria Paula, Muchagato Mauricio, Elisabete, Capelo-Martínez, José Luis, Lodeiro, Carlos, Valpuesta, José M., and Guerrero-Martínez, Andrés
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GOLD coatings ,NANORODS ,MESOPOROUS silica ,METHYLENE blue ,STAPHYLOCOCCUS aureus ,ESCHERICHIA coli ,TREHALOSE - Abstract
In this work, the successful preparation and characterization of gold nanorods (AuNRs) coated with a mesoporous silica shell (AuNRs@Simes) was achieved. Conjugation with methylene blue (MB) as a model drug using ultrasound-stimulated loading has been explored for further application in light-mediated antibacterial studies. Lyophilization of this conjugated nanosystem was analyzed using trehalose (TRH) as a cryogenic protector. The obtained stable dry formulation shows potent antimicrobial activity against Gram-negative (Escherichia coli) and Gram-positive (Staphylococcus aureus) bacteria after a simple post-treatment irradiation method with a red laser during a short time period. [ABSTRACT FROM AUTHOR]
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- 2021
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163. 3D reconstruction of the ATP-bound form of CCT reveals the asymmetric folding conformation of a type II chaperonin.
- Author
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Llorca, Oscar, Smyth, Martin G., Carrascosa, José L., Willison, Keith R., Radermacher, Michael, Steinbacher, Stefan, and Valpuesta, José M.
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MOLECULAR chaperones ,CYTOSOL ,ELECTRON microscopy ,ADENOSINE triphosphate - Abstract
The type II chaperonin CCT (chaperonin containing Tcp-1) of eukaryotic cytosol is a heteromeric 16-mer particle composed of eight different subunits. Three-dimensional reconstructions of apo-CCT and ATP-CCT have been obtained at 28 Å resolution by cryo-electron microscopy. Binding of ATP generates an asymmetric particle; one ring has a slightly different conformation from the apo-CCT ring, while the other has undergone substantial movements in the apical domains. Upon ATP binding the apical domains rotate and point towards the cylinder axis, so that the helical protrusions present at their tips could act as a lid closing the ring cavity. [ABSTRACT FROM AUTHOR]
- Published
- 1999
164. An intrinsic-tryptophan-fluorescence study of phage Φ29 connector/nucleic acid interactions.
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Urbaneja, Maíra A., Rivas, Susana, Carrascosa, José L., and Valpuesta, José M.
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FLUORESCENCE ,NUCLEIC acids ,TRYPTOPHAN ,BACTERIOPHAGES ,AMINO acids ,VIRUSES - Abstract
The protein p10 of bacteriophage Φ 29 assembled into connectors exhibit an intrinsic fluorescence with an emission peak centered at 335 nm, which suggests a hydrophobic environment of the three tryptohan residues that the protein contains. Upon incubation with linear DNA (but not with circular DNA), a decrease in the connector intrinsic fluorescence is measured which does not show any sequence specificity. The decrease in fluorescence is not observed when DNA is incubated with proteolyzed connectors, which lack the DNA-binding domain, suggesting that the fluorescence quenching is related to the binding of DNA to the Φ 29 connectors. Acrylamide quenching studies reveal a higher accessibility of tryptophan residues to the quencher when the connector is bound to DNA. Protein denaturation by guanidine hydrochloride occurs at lower denaturant concentrations in the presence of linear DNA (but not circular DNA) than in its absence, suggesting a conformational change of Φ 29 connector upon binding to linear DNA. This hypothesis is supported by the fact that the proteolyzed connectors, which do not bind DNA, are denatured at the same denaturant concentration, regardless of the presence of DNA. Φ 29 connectors also bind RNA, but this interaction does not exert any effect on acrylamide quenching or guanidine hydrochloride denaturation. This result, together with that showing that proteolyzed connectors are able to interact with RNA, reinforces the idea that Φ 29 connectors have two independent domains for interaction with DNA and RNA. [ABSTRACT FROM AUTHOR]
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- 1994
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165. Infrared spectroscopic studies of detergent-solubilized uncoupling protein from brown-adipose-tissue mitochondria.
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Rial, Eduardo, Muga, Arturo, Valpuesta, José M., Arrondo, José-Luis R., and Goñi, Félix M.
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PROTEINS ,BROWN adipose tissue ,MITOCHONDRIA ,MICELLES ,LIPIDS ,SURFACE active agents ,INFRARED spectroscopy - Abstract
The uncoupling protein of brown-adipose-tissue mitochondria has been purified in the form of mixed micelles with lipid and reduced Triton X-100. This surfactant has the advantage over conventional Triton X-100, that it does not interfere with amide bands in infrared spectra. The structure of the uncoupling protein in micellar form has been examined by Fourier-transform infrared spectroscopy (FTIR). In order to decompose the amide I contour into its components, band-narrowing (Fourier derivation and deconvolution) and band-decomposition techniques have been used. Combining data from spectra taken in H
2 O and ²H2 0 media, the following percentage distribution of secondary structure patterns has been obtained: 50% α-helix, 28–30% β-structure; 13–15% β-turns and 7% unordered. Thermal denaturation of the uncoupling protein has also been monitored by FTIR. In acordance with previous observations of different proteins, thermal denaturation is marked by a shift in the amide l maximum and the appearance of two new peaks in ²H2 0, at around 1620 cm-1 and 1685 cm-1 . Denaturation occurs in the 40–50°C temperature range, in agreement with studies of GDP-binding capacity. Cooling down the thermally denatured protein produces a new change in its secondary structure; however, the original conformation is not restored. The uncoupling protein possesses a nucleotide-binding site. On addition of GDP, small changes in protein conformation occur, attributable to changes in tertiary structure. However. no detectable effects are seen in the presence or absence of the other physiological regulators, the free fatty acids. The uncoupling protein shares important similarities in its primary structure with other anion carriers of the mitochondrial membrane; one of these, the adenine-nucleotide translocator, has been used in a comparative study, applying the same FTIR techniques described above for the uncoupling protein. Both proteins have a similar proportion of α-helix, probably corresponding to t he segments spanning the membrane, but the conformation of the polar domains appears to differ. [ABSTRACT FROM AUTHOR]- Published
- 1990
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166. Structural mechanism for tyrosine hydroxylase inhibition by dopamine and reactivation by Ser40 phosphorylation
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María Teresa Bueno-Carrasco, Jorge Cuéllar, Marte I. Flydal, César Santiago, Trond-André Kråkenes, Rune Kleppe, José R. López-Blanco, Miguel Marcilla, Knut Teigen, Sara Alvira, Pablo Chacón, Aurora Martinez, José M. Valpuesta, Ministerio de Ciencia e Innovación (España), Research Council of Norway, Western Norway Regional Health Authority, Bueno-Carrasco, M. Teresa, Cuéllar, Jorge, Flydal, Marte I., Santiago, César, Kråkenes, Trond-André, López-Blanco, José R., Marcilla, Miguel, Teigen, Knut, Alvira, Sara, Chacón, Pablo, Martinez, Aurora, Valpuesta, José M., Bueno-Carrasco, María Teresa [0000-0003-1586-2589], Cuéllar, Jorge [0000-0002-7789-807X], Flydal, Marte I. [0000-0002-4070-8367], Santiago, César [0000-0002-5149-1722], Kråkenes, Trond-André [0000-0001-8529-8448], López-Blanco, José R. [0000-0002-5891-4134], Marcilla, Miguel [0000-0001-9171-5076], Teigen, Knut [0000-0002-7031-9215], Alvira, Sara [0000-0003-3323-3436], Chacón, Pablo [0000-0002-3168-4826], Martinez, Aurora [0000-0003-1643-6506], and Valpuesta, José M. [0000-0001-7468-8053]
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Models, Molecular ,Multidisciplinary ,Tyrosine 3-Monooxygenase ,Science ,Dopamine ,Cryoelectron Microscopy ,General Physics and Astronomy ,General Chemistry ,Molecular neuroscience ,General Biochemistry, Genetics and Molecular Biology ,Article ,Catecholamines ,Protein Domains ,Catalytic Domain ,Humans ,Amino Acid Sequence ,Enzyme Inhibitors ,Phosphorylation ,Protein Binding - Abstract
16 pags, 7 figs . -- The online version contains supplementary movie1: https://static-content.springer.com/esm/art%3A10.1038%2Fs41467-021-27657-y/MediaObjects/41467_2021_27657_MOESM3_ESM.mp4, Tyrosine hydroxylase (TH) catalyzes the rate-limiting step in the biosynthesis of dopamine (DA) and other catecholamines, and its dysfunction leads to DA deficiency and parkinsonisms. Inhibition by catecholamines and reactivation by S40 phosphorylation are key regulatory mechanisms of TH activity and conformational stability. We used Cryo-EM to determine the structures of full-length human TH without and with DA, and the structure of S40 phosphorylated TH, complemented with biophysical and biochemical characterizations and molecular dynamics simulations. TH presents a tetrameric structure with dimerized regulatory domains that are separated 15 Å from the catalytic domains. Upon DA binding, a 20-residue α-helix in the flexible N-terminal tail of the regulatory domain is fixed in the active site, blocking it, while S40-phosphorylation forces its egress. The structures reveal the molecular basis of the inhibitory and stabilizing effects of DA and its counteraction by S40-phosphorylation, key regulatory mechanisms for homeostasis of DA and TH., This research was supported by the grant PID2019-105872GB-I00/AEI/10.13039/ 501100011033 from the Spanish Ministry of Science and Innovation to J.M.V. and J.C. as well as FRIMEDBIO (261826) from the Research Council of Norway to A.M.; the Western Norway Regional Health Authorities (912246 to A.M. and 912264 to R.K.), the K.G.
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- 2022
167. Assisted assembly of bacteriophage T7 core components for genome translocation across the bacterial envelope
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Mar Pérez-Ruiz, Juan Román Luque-Ortega, José M. Valpuesta, Ana Maria Cuervo, Mar Pulido-Cid, José L. Carrascosa, Ministerio de Ciencia, Innovación y Universidades (España), Pérez-Ruiz, Mar, Pulido-Cid, Mar, Luque-Ortega, Juan Román, Valpuesta, José M., Cuervo, Ana, Carrascosa, José L., Pérez-Ruiz, Mar [0000-0001-5776-1650], Pulido-Cid, Mar [0000-0002-5850-9710], Luque-Ortega, Juan Román [0000-0003-3206-7480], Valpuesta, José M. [0000-0001-7468-8053], Cuervo, Ana [0000-0001-9414-503X], and Carrascosa, José L. [0000-0002-4749-3522]
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Gene Expression Regulation, Viral ,Models, Molecular ,Protein Conformation ,Random hexamer ,Translocation, Genetic ,Morpholinos ,Bacteriophage ,chemistry.chemical_compound ,Podoviridae ,Protein structure ,Bacteriophage T7 ,Image Processing, Computer-Assisted ,Amino Acid Sequence ,Cryo-EM ,Multidisciplinary ,biology ,Viral Core Proteins ,Cryoelectron Microscopy ,DNA translocation ,Periplasmic space ,Transglycosylase ,Virus Internalization ,Biological Sciences ,biology.organism_classification ,Microscopy, Electron ,chemistry ,DNA, Viral ,Biophysics ,Peptidoglycan ,Cell envelope ,DNA - Abstract
28 p.-5 fig.- + 4 fig. supl.-1 tab. supl., In most bacteriophages, genome transport across bacterial envelopes is carried out by the tail machinery. In viruses of the Podoviridae family, in which the tail is not long enough to traverse the bacterial wall, it has been postulated that viral core proteins assembled inside the viral head are translocated and reassembled into a tube within the periplasm that extends the tail channel. Bacteriophage T7 infects Escherichia coli, and despite extensive studies, the precise mechanism by which its genome is translocated remains unknown. Using cryo-electron microscopy, we have resolved the structure of two different assemblies of the T7 DNA translocation complex composed of the core proteins gp15 and gp16. Gp15 alone forms a partially folded hexamer, which is further assembled upon interaction with gp16 into a tubular structure, forming a channel that could allow DNA passage. The structure of the gp15–gp16 complex also shows the location within gp16 of a canonical transglycosylase motif involved in the degradation of the bacterial peptidoglycan layer. This complex docks well in the tail extension structure found in the periplasm of T7-infected bacteria and matches the sixfold symmetry of the phage tail. In such cases, gp15 and gp16 that are initially present in the T7 capsid eightfold-symmetric core would change their oligomeric state upon reassembly in the periplasm. Altogether, these results allow us to propose a model for the assembly of the core translocation complex in the periplasm, which furthers understanding of the molecular mechanism involved in the release of T7 viral DNA into the bacterial cytoplasm., This work was supported by the Ministry of Science,Innovation and Universities of Spain, Grant BFU 2014-54181 (to J.L.C.) and Contracts SEV-2013-0347 (to A.C.) and BES-2015-073615 to (M.P.-R.).
- Published
- 2021
168. DNA conformational change induced by the bacteriophage {Phi}29 connector
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Valpuesta, José M., Serrano, Manuel, Donate, Luis E., Herranz, Lucía, and Carrascosa, José L.
- Abstract
Translocation of viral DNA inwards and outwards of the capsid of double-stranded DNA bacteriophages occurs through the connector, a key viral structure that is known to interact with DNA. It is shown here that phage Φ29 connector binds both linear and circular double-stranded DNA. However, DNA-mediated protection of Φ29 connectors against Staphylococcus aureus endoprotease V8 digestion suggests that binding to linear DNA is more stable than to circular DNA. Endoprotease V8-protection assays also suggest that the length of the linear DNA required to produce a stable Φ29 connector-DNA interaction is, at least, twice longer than the Φ29 connector channel. This result is confirmed by experiments of Φ29 connector-protection of DNA against DNase I digestion. Furthermore, DNA circularization assays indicate that Φ29 connectors restrain negative supercoiling when bound to linear DNA. This DNA conformational change is not observed upon binding to circular DNA and it could reflect the existence of some left-handed DNA coiling or DNA untwisting inside of the Φ29 connector channel.
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- 1992
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169. Structural insights into the ability of nucleoplasmin to assemble and chaperone histone octamers for DNA deposition
- Author
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Rocío Arranz, Aitor Franco, Adelina Prado, Jaime Martín-Benito, Adrián Velázquez-Campoy, Arturo Muga, Noelia Fernández-Rivero, José M. Valpuesta, Joan Segura, Valpuesta, José M., Martín-Benito, Jaime, Segura, Joan, Valpuesta, José M. [0000-0001-7468-8053], Martín-Benito, Jaime [0000-0002-8541-4709], and Segura, Joan [0000-0001-5593-735X]
- Subjects
0301 basic medicine ,Nucleoplasmin ,lcsh:Medicine ,mechanism ,acidic protein ,Xenopus Proteins ,sperm chromatin ,Article ,Avian Proteins ,Histones ,03 medical and health sciences ,chemistry.chemical_compound ,Xenopus laevis ,0302 clinical medicine ,chromatin decondensation ,Cryoelectron microscopy ,Biophysical chemistry ,Nucleosome ,Animals ,Histone octamer ,lcsh:Science ,education ,Nucleoplasmins ,education.field_of_study ,complexes ,Multidisciplinary ,biology ,tetramer ,lcsh:R ,nucleosome ,crystal-structure ,core ,DNA ,In vitro ,Chromatin ,Nucleosomes ,030104 developmental biology ,Histone ,chemistry ,Chaperone (protein) ,Biophysics ,biology.protein ,lcsh:Q ,transcription ,Chickens ,030217 neurology & neurosurgery - Abstract
Nucleoplasmin (Np) is a pentameric histone chaperone that regulates the condensation state of chromatin in different cellular processes. We focus here on the interaction of NP with the histone octamer, showing that NP could bind sequentially the histone components to assemble an octamer-like particle, and crosslinked octamers with high affinity. The three-dimensional reconstruction of the Np/octamer complex generated by single-particle cryoelectron microscopy, revealed that several intrinsically disordered tail domains of two NP pentamers, facing each other through their distal face, encage the histone octamer in a nucleosome-like conformation and prevent its dissociation. Formation of this complex depended on post-translational modification and exposure of the acidic tract at the tail domain of NP. Finally, NP was capable of transferring the histone octamers to DNA in vitro, assembling nucleosomes. This activity may have biological relevance for processes in which the histone octamer must be rapidly removed from or deposited onto the DNA., This work was supported by Agencia Española de Investigación/Fondos de Desarrollo Regional (AEI/FEDER, UE), [BFU2016-75984 to J.M.V., BFU2016-75983 to A.M.] and the Basque Government [IT709-13 to A.M.]
- Published
- 2019
170. Engineering protein assemblies with allosteric control via monomer fold-switching
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Beatriz Ibarra-Molero, Luis A. Campos, Germán Rivas, Jose M. Sanchez-Ruiz, Sara Alvira, Antonio A. Romero, Carlos Alfonso, Victor Muñoz, José M. Valpuesta, Rajendra Sharma, Federico M. Ruiz, Mourad Sadqi, European Research Council, Ministerio de Economía y Competitividad (España), Sharma, Rajendra, Sadqi, Mourad, Alfonso, Carlos, Romero Garrido, Antonio, Valpuesta, José M., Muñoz, Víctor, Sharma, Rajendra [0000-0003-0516-3258], Sadqi, Mourad [0000-0003-3553-3408], Alfonso, Carlos [0000-0001-7165-4800], Romero Garrido, Antonio [0000-0002-6990-6973], Valpuesta, José M. [0000-0001-7468-8053], and Muñoz, Víctor [0000-0002-5683-1482]
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0301 basic medicine ,Protein Folding ,Serine Proteinase Inhibitors ,Fold (higher-order function) ,Science ,Allosteric regulation ,General Physics and Astronomy ,Molecular Dynamics Simulation ,Protein Engineering ,010402 general chemistry ,01 natural sciences ,Protein Structure, Secondary ,Article ,General Biochemistry, Genetics and Molecular Biology ,Molecular engineering ,Computational biophysics ,03 medical and health sciences ,chemistry.chemical_compound ,Allosteric Regulation ,Nanobiotechnology ,Protein folding ,Cloning, Molecular ,lcsh:Science ,Multidisciplinary ,Chemistry ,Proteins ,General Chemistry ,Recombinant Proteins ,0104 chemical sciences ,3. Good health ,Molecular Docking Simulation ,ComputingMethodologies_PATTERNRECOGNITION ,030104 developmental biology ,Monomer ,Structural biology ,Mutation ,Biophysics ,lcsh:Q ,Protein Multimerization ,Serine Proteases - Abstract
The macromolecular machines of life use allosteric control to self-assemble, dissociate and change shape in response to signals. Despite enormous interest, the design of nanoscale allosteric assemblies has proven tremendously challenging. Here we present a proof of concept of allosteric assembly in which an engineered fold switch on the protein monomer triggers or blocks assembly. Our design is based on the hyper-stable, naturally monomeric protein CI2, a paradigm of simple two-state folding, and the toroidal arrangement with 6-fold symmetry that it only adopts in crystalline form. We engineer CI2 to enable a switch between the native and an alternate, latent fold that self-assembles onto hexagonal toroidal particles by exposing a favorable inter-monomer interface. The assembly is controlled on demand via the competing effects of temperature and a designed short peptide. These findings unveil a remarkable potential for structural metamorphosis in proteins and demonstrate key principles for engineering protein-based nanomachinery., European Research Council (grant ERC-2012-ADG-323059 to V.M.) and by the PRODESTECH network funded through the CONSOLIDER program from the Spanish Government (grant CSD2009-00088). L.A.C. acknowledges support from Ministry of Economy and Competitiveness through grants BIO2016-78768-P and RYC-2013-13197.
- Published
- 2019
171. Nucleus-translocated mitochondrial cytochrome c liberates nucleophosmin-sequestered ARF tumor suppressor by changing nucleolar liquid-liquid phase separation
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Katiuska González-Arzola, Antonio Díaz-Quintana, Noelia Bernardo-García, Jonathan Martínez-Fábregas, Francisco Rivero-Rodríguez, Miguel Á. Casado-Combreras, Carlos A. Elena-Real, Alejandro Velázquez-Cruz, Sergio Gil-Caballero, Adrián Velázquez-Campoy, Elzbieta Szulc, María P. Gavilán, Isabel Ayala, Rocío Arranz, Rosa M. Ríos, Xavier Salvatella, José M. Valpuesta, Juan A. Hermoso, Miguel A. De la Rosa, Irene Díaz-Moreno, Scientific Research Centre 'Isla de la Cartuja' (cicCartuja), Universidad de Sevilla / University of Sevilla, Université Paris-Saclay, Institut National de Recherche pour l’Agriculture, l’Alimentation et l’Environnement (INRAE), University of Dundee, Institut de biologie structurale (IBS - UMR 5075 ), Centre National de la Recherche Scientifique (CNRS)-Université Grenoble Alpes [2016-2019] (UGA [2016-2019])-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA), Unidad Asociada IQFR-CSIC-BIFI [Zaragoza, Spain], University of Zaragoza - Universidad de Zaragoza [Zaragoza]-Instituto de Biocomputación y Física de Sistemas Complejos - BIFI [Zaragoza, Spain], The Barcelona Institute of Science and Technology, Centro Andaluz de Biología Molecular y Medicina Regenerativa (CABIMER), Institut de biologie structurale (IBS - UMR 5075), Centre National de la Recherche Scientifique (CNRS)-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Grenoble Alpes (UGA), Centro Nacional de Biotecnología [Madrid] (CNB-CSIC), Consejo Superior de Investigaciones Científicas [Madrid] (CSIC), Consejo Superior de Investigaciones Científicas (CSIC), Institute of Physical Chemistry Rocasolano (IQFR), Instituto de Investigaciones Químicas (IIQ), Universidad de Sevilla / University of Sevilla-Consejo Superior de Investigaciones Científicas [Madrid] (CSIC), Horizon 2020 program of the European Commission,Cámara Foundation (C.A.E.R.´s fellowship), European Regional Development Fund Regional Development Fund (FEDER), European Project: 648201,H2020,ERC-2014-CoG,CONCERT(2015), Universidad de Sevilla, ALBA Synchrotron, Ministerio de Economía y Competitividad (España), Ministerio de Ciencia e Innovación (España), Junta de Andalucía, European Commission, Fundación Ramón Areces, Ministerio de Educación, Cultura y Deporte (España), Fundación 'la Caixa', Fundación Científica Asociación Española Contra el Cáncer, González-Arzola, Katiuska, Díaz-Quintana, Antonio, Martínez-Fábregas, Jonathan, Casado-Combreras, Miguel Á., Velázquez-Cruz, Alejandro, Velázquez-Campoy, Adrián, Gavilán, María P., Arranz, Rocío, Salvatella, Xavier, Valpuesta, José M., Hermoso, Juan A., Rosa, Miguel A. de la, and Díaz-Moreno, Irene
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MESH: Caspases ,[SDV.BBM.BS]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Structural Biology [q-bio.BM] ,MESH: Mitochondria ,Lysine ,Tumor Suppressor Proteins ,MESH: Arginine ,Cytochromes c ,Nuclear Proteins ,MESH: Nucleophosmin ,MESH: Cytochromes c ,Arginine ,Mitochondria ,Structural Biology ,Caspases ,MESH: Lysine ,MESH: Tumor Suppressor Proteins ,Molecular Biology ,MESH: Nuclear Proteins ,Nucleophosmin - Abstract
32 pags., 17 figs., 1 tab., The regular functioning of the nucleolus and nucleus-mitochondria crosstalk are considered unrelated processes, yet cytochrome c (Cc) migrates to the nucleus and even the nucleolus under stress conditions. Nucleolar liquid-liquid phase separation usually serves the cell as a fast, smart mechanism to control the spatial localization and trafficking of nuclear proteins. Actually, the alternative reading frame (ARF), a tumor suppressor protein sequestered by nucleophosmin (NPM) in the nucleoli, is shifted out from NPM upon DNA damage. DNA damage also triggers early translocation of respiratory Cc to nucleus before cytoplasmic caspase activation. Here, we show that Cc can bind to nucleolar NPM by triggering an extended-to-compact conformational change, driving ARF release. Such a NPM-Cc nucleolar interaction can be extended to a general mechanism for DNA damage in which the lysine-rich regions of Cc-rather than the canonical, arginine-rich stretches of membrane-less organelle components-controls the trafficking and availability of nucleolar proteins., We thank the staf at the NMR facility at CITIUS (University of Seville), the microscopy facility at CABIMER (Seville) and the ALBA Synchrotron (Barcelona). We are grateful to the Spanish Government (grant nos. PID2021-126663NB-I00, PGC2018-096049-B-I00/FEDER, BIO2015-70092-R, BFU2015-71017/BMC, BFU2016-75984/BMC, PID2019-105872GB and BFU2017-90030-P, FEDER/Ministerio de Ciencia e Innovación–Agencia Estatal de Investigación), European Regional Development Fund (FEDER), the Regional Government of Andalusia (grant nos. BIO-198; US-1254317, US-1257019, P18-FR-3487 and P18-HO-4091, US/JUNTA/FEDER, UE), the European Commission: European Regional Development Fund and European Research Council (CONCERT, contract number 648201) and the Ramón Areces Foundation. This work has been supported by Infrastructure for NMR, EM and X-rays for Translational Research (iNEXT, grant no. PID 3407) funded by the Horizon 2020 program of the European Commission, Cámara Foundation (C.A.E.R.´s fellowship), the Spanish Ministry of Education, Culture and Sports (grant nos. FPU18/06577, FPU16/01513 and FPU013/04373; M.A.C.-C., A.V.-C. and F.R.-R.’s fellowships, respectively), La Caixa Foundation (E.S.’s fellowship), Severo Ochoa Award of Excellence from MINECO (Government of Spain, IRB Barcelona and CNB Madrid) and the Spanish Association Against Cancer Scientific Foundation (FC AECC, M.P.G.’s postdoctoral grant)
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- 2021
172. The Structure of Native Influenza Virion Ribonucleoproteins.
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Arranz, Racío, Coloma, Racío, Chichón, Francisco Javier, Conesa, José Javier, Carroscasa, José L., Valpuesta, José M., Ortín, Juan, and Martín-Benito, Jaime
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NUCLEOPROTEIN structure , *INFLUENZA A virus , *VIRAL genomes , *VIRION , *MOLECULAR structure of RNA , *RNA polymerases , *MOLECULAR conformation , *ELECTRON microscopy , *TOMOGRAPHY , *DOUBLE helix structure , *VIRAL replication - Abstract
The article discusses the structural determination of native ribonucleoproteins of the influenza A virus genome, which are composed of negative-stranded RNAs , polymerase complex, and nucleoproteins (NPs). Reconstruction of virion RNP structure with cryo-electron microscopy and cryoelectron tomography yielded a double-helical conformation with opposite polarity NP strands connected by a loop. Other topics include RNP replication and genetic transcription, nuclear import of RNPs of the parent virus, and RNP encapsidation in progeny virions.
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- 2012
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173. Active DNA unwinding dynamics during processive DNA replication.
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Morin, José A., Cao, Francisco J., Lázaro, José M., Arias-GonzaIez, J. Ricardo, Valpuesta, José M., Carrascosa, José L., Salas, Margarita, and Ibarra, Borja
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DNA , *DNA replication , *DNA polymerases , *DNA synthesis , *NUCLEIC acids - Abstract
Duplication of double-stranded DNA (dsDNA) requires a fine-tuned coordination between the DNA replication and unwinding reactions. Using optical tweezers, we probed the coupling dynamics between these two activities when they are simultaneously carried out by individual Phi29 DNA polymerase molecules replicating a dsDNA hairpin. We used the wild-type and an unwinding deficient polymerase variant and found that mechanical tension applied on the DNA and the DNA sequence modulate in different ways the replication, unwinding rates, and pause kinetics of each polymerase. However, incorporation of pause kinetics in a model to quantify the unwinding reaction reveals that both polymerases destabilize the fork with the same active mechanism and offers insights into the topological strategies that could be used by the Phi29 DNA polymerase and other DNA replication systems to couple unwinding and replication reactions. [ABSTRACT FROM AUTHOR]
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- 2012
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174. Characterization of the structure and self-recognition of the human centrosomal protein NA14: implications for stability and function.
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Rodríguez-Rodríguez, Mar, Treviño, Miguel A., Laurents, Douglas V., Arranz, Rocío, Valpuesta, José M., Rico, Manuel, Bruix, Marta, and Jiménez, M.Angeles
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PROTEIN structure , *CELLULAR recognition , *MICROTUBULES , *CIRCULAR dichroism , *NUCLEAR magnetic resonance spectroscopy , *GENETIC mutation , *PH effect - Abstract
The protein NA14 is a key adaptor protein mediating the intermolecular interactions of microtubules and Spastin. To gain insight into its structure and function, we have expressed, purified and characterized human NA14 and some variants. NA14 is rather insoluble and tends to oligomerize and form fibrils. Successive mutation of the three Cys and two potentially exposed Leu residues (83 and 93) yielded a water-soluble quintuple variant, named 3CS-2LR. NA14 and its variants have a high helical content as determined by circular dichroism (CD). Based on nuclear magnetic resonance data of the quintuple mutant and the wild-type (wt) protein in the presence of dodecylphosphocholine micelles, the N-(M1-N13) and C-termini (K105-S119) were found to lack preferred structure. The remaining residues (14–104) participate in NA14 self-association, probably by forming a parallel coiled-coil structure. We hypothesize that Leu 83 and Leu 93 mediate interactions among NA14, Spastin and microtubules. We have also examined urea and thermal denaturation of the quintuple and other NA14 variants at different pH values by CD. The pH dependence of the conformational stability and the elevated native-state pKa determined for the two conserved Tyr allow us to propose that the NA14 structure may be stabilized by two Glu-COO− ||| HO-Tyr H-bonds, highly conserved in NA14-like proteins in other species. [ABSTRACT FROM AUTHOR]
- Published
- 2011
175. Truncation-Driven Lateral Association of α-Synuclein Hinders Amyloid Clearance by the Hsp70-Based Disaggregase.
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Franco, Aitor, Cuéllar, Jorge, Fernández-Higuero, José Ángel, de la Arada, Igor, Orozco, Natalia, Valpuesta, José M., Prado, Adelina, and Muga, Arturo
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AMYLOID , *CALPAIN , *POST-translational modification , *NEURODEGENERATION , *QUALITY control , *DEPOLYMERIZATION - Abstract
The aggregation of α-synuclein is the hallmark of a collective of neurodegenerative disorders known as synucleinopathies. The tendency to aggregate of this protein, the toxicity of its aggregation intermediates and the ability of the cellular protein quality control system to clear these intermediates seems to be regulated, among other factors, by post-translational modifications (PTMs). Among these modifications, we consider herein proteolysis at both the N- and C-terminal regions of α-synuclein as a factor that could modulate disassembly of toxic amyloids by the human disaggregase, a combination of the chaperones Hsc70, DnaJB1 and Apg2. We find that, in contrast to aggregates of the protein lacking the N-terminus, which can be solubilized as efficiently as those of the WT protein, the deletion of the C-terminal domain, either in a recombinant context or as a consequence of calpain treatment, impaired Hsc70-mediated amyloid disassembly. Progressive removal of the negative charges at the C-terminal region induces lateral association of fibrils and type B* oligomers, precluding chaperone action. We propose that truncation-driven aggregate clumping impairs the mechanical action of chaperones, which includes fast protofilament unzipping coupled to depolymerization. Inhibition of the chaperone-mediated clearance of C-truncated species could explain their exacerbated toxicity and higher propensity to deposit found in vivo. [ABSTRACT FROM AUTHOR]
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- 2021
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176. All three chaperonin genes in the archaeon Haloferax volcanii are individually dispensable.
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Kapatai, Georgia, Large, Andrew, Benesch, Justin L. P., Robinson, Carol V., Carrascosa, José L., Valpuesta, José M., Gowrinathan, Preethy, and Lund, Peter A.
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MOLECULAR chaperones , *PHYLOGENY , *BACTERIA , *MITOCHONDRIA , *CHLOROPLASTS , *GENES - Abstract
The Hsp60 or chaperonin class of molecular chaperones is divided into two phylogenetic groups: group I, found in bacteria, mitochondria and chloroplasts, and group II, found in eukaryotic cytosol and archaea. Group I chaperonins are generally essential in bacteria, although when multiple copies are found one or more of these are dispensable. Eukaryotes contain eight genes for group II chaperonins, all of which are essential, and it has been shown that these proteins assemble into double-ring complexes with eightfold symmetry where all proteins occupy specific positions in the ring. In archaea, there are one, two or three genes for the group II chaperonins, but whether they are essential for growth is unknown. Here we describe a detailed genetic, structural and biochemical analysis of these proteins in the halophilic archaeon, Haloferax volcanii. This organism contains three genes for group II chaperonins, and we show that all are individually dispensable but at least one must be present for growth. Two of the three possible double mutants can be constructed, but only one of the three genes is capable of fully complementing the stress-dependent phenotypes that these double mutants show. The chaperonin complexes are made up of hetero-oligomers with eightfold symmetry, and the properties of the different combinations of subunits derived from the mutants are distinct. We conclude that, although they are more homologous to eukaryotic than prokaryotic chaperonins, archaeal chaperonins have some redundancy of function. [ABSTRACT FROM AUTHOR]
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- 2006
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177. Structure of the complex between the cytosolic chaperonin CCI and phosducin-like protein.
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Martin-Benito, Jaime, Bertrand, Sara, Ting Hu, Ludtke, Paul J., Mclaughlin, Joseph N., Wiflardson, Barry M., Carrascosa, José L., and Valpuesta, José M.
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MOLECULAR chaperones , *PROTEINS , *CRYOELECTRONICS , *MICROSCOPY , *ATOMIC structure , *PROTEIN binding - Abstract
The three-dimensional structure of the complex formed between the cytosolic chaperonin CCT (chaperonin containing TCP-1) and phosducin (Pdc)-like protein (PhLP), a regulator of cc activity, has been solved by cryoelectron microscopy. Binding of PhLP to cc occurs through only one of the chaperonin rings, and the protein does not occupy the central folding cavity but rather sits above it through interactions with two regions on opposite sides of the ring. This causes the apical domains of the CCT subunits to close in, thus excluding access to the folding cavity. The atomic model of PhLP generated from several atomic structures of the homologous Pdc fits very well with the mass of the complex attributable to PhIP and predicts the involvement of several sequences of PhLP in CCT binding. Binding experiments performed with PhLP/Pdc chimeric proteins, taking advantage of the fact that Pdc does not interact with CCT, confirm that both the N- and C-terminal domains of PhLP are involved in CCT binding and that several regions suggested by the docking experiment are indeed critical in the interaction with the cytosolic chaperonin. [ABSTRACT FROM AUTHOR]
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- 2004
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178. Molecular clamp mechanism of substrate binding by hydrophobic coiled-coil residues of the archaeal chaperone prefoldin.
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Lundin, Victor F., Stirling, Peter C., Gomez-Reino, Juan, Mwenifumbo, Jill C., Obst, Jennifer M., Valpuesta, José M., and Leroux, Michel R.
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MOLECULAR chaperones , *ACTIN , *TUBULINS , *PROTEINS , *ORIGIN of life , *CHIMERAS (Botany) - Abstract
Prefoldin (PFD) is a jellyfish-shaped molecular chaperone that has been proposed to play a general role in de novo protein folding in archaea and is known to assist the biogenesis of actins, tubulins, and potentially other proteins in eukaryotes. Using point mutants, chimeras, and intradomain swap variants, we show that the six coiled-coil tentacles of archaeal PFD act in concert to bind and stabilize nonnative proteins near the opening of the cavity they form. Importantly, the interaction between chaperone and substrate depends on the mostly buried interhelical hydrophobic residues of the coiled coils. We also show by electron microscopy that the tentacles can undergo an en bloc movement to accommodate an unfolded substrate. Our data reveal how archael PFD uses its unique architecture and intrinsic coiled-coil properties to interact with nonnative polypeptides. [ABSTRACT FROM AUTHOR]
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- 2004
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179. 3D structure of the influenza virus polymerase complex: Localization of subunit domains.
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Area, Estela, Martin-Benito, Jaime, Gastaminza, Pablo, Torreira, Eva, Valpuesta, José M., Carrascosa, José L., and Ortin, Juan
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IMMUNOCHEMISTRY , *INFLUENZA viruses , *MULTIENZYME complexes , *NUCLEOPROTEINS , *DNA viruses , *RNA viruses , *ELECTRON microscopy - Abstract
The 3D structure of the influenza virus polymerase complex was determined by electron microscopy and image processing of recombinant ribonucleoproteins (RNPs). The RNPs were generated by in vivo amplification using cDNAs of the three polymerase subunits, the nucleoprotein, and a model virus-associated RNA containing 248 nt. The polymerase structure obtained is very compact, with no apparent boundaries among subunits. The position of specific regions of the PB1, PB2, and PA subunits was determined by 3D reconstruction of either RNP-mAb complexes or tagged RNPs. This structural model is available for the polymerase of a negative-stranded RNA virus and provides a general delineation of the complex and its interaction with the template-associated nucleoprotein monomers in the RNP. [ABSTRACT FROM AUTHOR]
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- 2004
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180. Globular Aggregates Stemming from the Self-Assembly of an Amphiphilic N -Annulated Perylene Bisimide in Aqueous Media.
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Martínez, Manuel A., Greciano, Elisa E., Cuéllar, Jorge, Valpuesta, José M., and Sánchez, Luis
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HYDROPHOBIC interactions , *ETHYLENE glycol , *TRANSMISSION electron microscopy , *DIOXANE , *PERYLENE , *SPIROKETALS , *DENATURATION of proteins - Abstract
Herein, we describe the synthesis of highly emissive amphiphilic N-annulated PBI 1 decorated with oligo ethylene glycol (OEG) side chains. These polar side chains allow the straightforward solubility of 1 in solvents of different polarity such as water, iPrOH, dioxane, or chloroform. Compound 1 self-assembles in aqueous media by π-stacking of the aromatic units and van der Waals interactions, favored by the hydrophobic effect. The hypo- and hypsochromic effect observed in the UV-Vis spectra of 1 in water in comparison to chloroform is diagnostic of H-type aggregation. Solvent denaturation experiments allow deriving the free Gibbs energy for the self-assembly process in aqueous media and the factor m that is indicative of the influence exerted by a good solvent in the stability of the final aggregates. The ability of compound 1 to self-assemble in water yields globular aggregates that have been visualized by TEM imaging. [ABSTRACT FROM AUTHOR]
- Published
- 2021
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181. Structural insights into the ability of nucleoplasmin to assemble and chaperone histone octamers for DNA deposition.
- Author
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Franco, Aitor, Arranz, Rocío, Fernández-Rivero, Noelia, Velázquez-Campoy, Adrián, Martín-Benito, Jaime, Segura, Joan, Prado, Adelina, Valpuesta, José M., and Muga, Arturo
- Abstract
Nucleoplasmin (NP) is a pentameric histone chaperone that regulates the condensation state of chromatin in different cellular processes. We focus here on the interaction of NP with the histone octamer, showing that NP could bind sequentially the histone components to assemble an octamer-like particle, and crosslinked octamers with high affinity. The three-dimensional reconstruction of the NP/octamer complex generated by single-particle cryoelectron microscopy, revealed that several intrinsically disordered tail domains of two NP pentamers, facing each other through their distal face, encage the histone octamer in a nucleosome-like conformation and prevent its dissociation. Formation of this complex depended on post-translational modification and exposure of the acidic tract at the tail domain of NP. Finally, NP was capable of transferring the histone octamers to DNA in vitro, assembling nucleosomes. This activity may have biological relevance for processes in which the histone octamer must be rapidly removed from or deposited onto the DNA. [ABSTRACT FROM AUTHOR]
- Published
- 2019
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182. The chaperonin CCT inhibits assembly of α-synuclein amyloid fibrils by a specific, conformation-dependent interaction.
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Sot, Begoña, Rubio-Muñoz, Alejandra, Leal-Quintero, Ahudrey, Martínez-Sabando, Javier, Marcilla, Miguel, Roodveldt, Cintia, and Valpuesta, José M.
- Abstract
The eukaryotic chaperonin CCT (chaperonin containing TCP-1) uses cavities built into its double-ring structure to encapsulate and to assist folding of a large subset of proteins. CCT can inhibit amyloid fibre assembly and toxicity of the polyQ extended mutant of huntingtin, the protein responsible for Huntington's disease. This raises the possibility that CCT modulates other amyloidopathies, a still-unaddressed question. We show here that CCT inhibits amyloid fibre assembly of α-synuclein A53T, one of the mutants responsible for Parkinson's disease. We evaluated fibrillation blockade in α-synuclein A53T deletion mutants and CCT interactions of full-length A53T in distinct oligomeric states to define an inhibition mechanism specific for α-synuclein. CCT interferes with fibre assembly by interaction of its CCTζ and CCTγ subunits with the A53T central hydrophobic region (NAC). This interaction is specific to NAC conformation, as it is produced once soluble α-synuclein A53T oligomers form and blocks the reaction before fibres begin to grow. Finally, we show that this association inhibits α-synuclein A53T oligomer toxicity in neuroblastoma cells. In summary, our results and those for huntingtin suggest that CCT is a general modulator of amyloidogenesis via a specific mechanism. [ABSTRACT FROM AUTHOR]
- Published
- 2017
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183. Chaperonin CCT controls extracellular vesicle production and cell metabolism through kinesin dynamics.
- Author
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Rojas-Gómez A, Dosil SG, Chichón FJ, Fernández-Gallego N, Ferrarini A, Calvo E, Calzada-Fraile D, Requena S, Otón J, Serrano A, Tarifa R, Arroyo M, Sorrentino A, Pereiro E, Vázquez J, Valpuesta JM, Sánchez-Madrid F, and Martín-Cófreces NB
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- Chaperonin Containing TCP-1 metabolism, Lipid Metabolism, Lipids, Kinesins metabolism, Extracellular Vesicles metabolism
- Abstract
Cell proteostasis includes gene transcription, protein translation, folding of de novo proteins, post-translational modifications, secretion, degradation and recycling. By profiling the proteome of extracellular vesicles (EVs) from T cells, we have found the chaperonin complex CCT, involved in the correct folding of particular proteins. By limiting CCT cell-content by siRNA, cells undergo altered lipid composition and metabolic rewiring towards a lipid-dependent metabolism, with increased activity of peroxisomes and mitochondria. This is due to dysregulation of the dynamics of interorganelle contacts between lipid droplets, mitochondria, peroxisomes and the endolysosomal system. This process accelerates the biogenesis of multivesicular bodies leading to higher EV production through the dynamic regulation of microtubule-based kinesin motors. These findings connect proteostasis with lipid metabolism through an unexpected role of CCT., (© 2023 The Authors. Journal of Extracellular Vesicles published by Wiley Periodicals, LLC on behalf of the International Society for Extracellular Vesicles.)
- Published
- 2023
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184. Nucleus-translocated mitochondrial cytochrome c liberates nucleophosmin-sequestered ARF tumor suppressor by changing nucleolar liquid-liquid phase separation.
- Author
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González-Arzola K, Díaz-Quintana A, Bernardo-García N, Martínez-Fábregas J, Rivero-Rodríguez F, Casado-Combreras MÁ, Elena-Real CA, Velázquez-Cruz A, Gil-Caballero S, Velázquez-Campoy A, Szulc E, Gavilán MP, Ayala I, Arranz R, Ríos RM, Salvatella X, Valpuesta JM, Hermoso JA, De la Rosa MA, and Díaz-Moreno I
- Subjects
- Arginine, Caspases, Lysine, Mitochondria metabolism, Nuclear Proteins metabolism, Tumor Suppressor Proteins, Cytochromes c, Nucleophosmin
- Abstract
The regular functioning of the nucleolus and nucleus-mitochondria crosstalk are considered unrelated processes, yet cytochrome c (Cc) migrates to the nucleus and even the nucleolus under stress conditions. Nucleolar liquid-liquid phase separation usually serves the cell as a fast, smart mechanism to control the spatial localization and trafficking of nuclear proteins. Actually, the alternative reading frame (ARF), a tumor suppressor protein sequestered by nucleophosmin (NPM) in the nucleoli, is shifted out from NPM upon DNA damage. DNA damage also triggers early translocation of respiratory Cc to nucleus before cytoplasmic caspase activation. Here, we show that Cc can bind to nucleolar NPM by triggering an extended-to-compact conformational change, driving ARF release. Such a NPM-Cc nucleolar interaction can be extended to a general mechanism for DNA damage in which the lysine-rich regions of Cc-rather than the canonical, arginine-rich stretches of membrane-less organelle components-controls the trafficking and availability of nucleolar proteins., (© 2022. The Author(s), under exclusive licence to Springer Nature America, Inc.)
- Published
- 2022
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- View/download PDF
185. The Molecular Chaperone CCT Sequesters Gelsolin and Protects it from Cleavage by Caspase-3.
- Author
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Cuéllar J, Vallin J, Svanström A, Maestro-López M, Bueno-Carrasco MT, Ludlam WG, Willardson BM, Valpuesta JM, and Grantham J
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- Actins metabolism, Animals, Cryoelectron Microscopy, Mice, Caspase 3 metabolism, Chaperonin Containing TCP-1 metabolism, Gelsolin chemistry, Gelsolin metabolism, Proteolysis
- Abstract
The actin filament severing and capping protein gelsolin plays an important role in modulation of actin filament dynamics by influencing the number of actin filament ends. During apoptosis, gelsolin becomes constitutively active due to cleavage by caspase-3. In non-apoptotic cells gelsolin is activated by the binding of Ca
2+ . This activated form of gelsolin binds to, but is not a folding substrate of the molecular chaperone CCT/TRiC. Here we demonstrate that in vitro, gelsolin is protected from cleavage by caspase-3 in the presence of CCT. Cryoelectron microscopy and single particle 3D reconstruction of the CCT:gelsolin complex reveals that gelsolin is located in the interior of the chaperonin cavity, with a placement distinct from that of the obligate CCT folding substrates actin and tubulin. In cultured mouse melanoma B16F1 cells, gelsolin co-localises with CCT upon stimulation of actin dynamics at peripheral regions during lamellipodia formation. These data indicate that localised sequestration of gelsolin by CCT may provide spatial control of actin filament dynamics., Competing Interests: Conflict of interest The authors declare no competing interests., (Copyright © 2021 The Authors. Published by Elsevier Ltd.. All rights reserved.)- Published
- 2022
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186. Structural mechanism for tyrosine hydroxylase inhibition by dopamine and reactivation by Ser40 phosphorylation.
- Author
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Bueno-Carrasco MT, Cuéllar J, Flydal MI, Santiago C, Kråkenes TA, Kleppe R, López-Blanco JR, Marcilla M, Teigen K, Alvira S, Chacón P, Martinez A, and Valpuesta JM
- Subjects
- Amino Acid Sequence, Catalytic Domain, Catecholamines metabolism, Cryoelectron Microscopy, Dopamine chemistry, Dopamine metabolism, Enzyme Inhibitors chemistry, Enzyme Inhibitors metabolism, Humans, Models, Molecular, Phosphorylation, Protein Binding, Protein Domains, Tyrosine 3-Monooxygenase genetics, Tyrosine 3-Monooxygenase metabolism, Dopamine pharmacology, Enzyme Inhibitors pharmacology, Tyrosine 3-Monooxygenase antagonists & inhibitors, Tyrosine 3-Monooxygenase chemistry
- Abstract
Tyrosine hydroxylase (TH) catalyzes the rate-limiting step in the biosynthesis of dopamine (DA) and other catecholamines, and its dysfunction leads to DA deficiency and parkinsonisms. Inhibition by catecholamines and reactivation by S40 phosphorylation are key regulatory mechanisms of TH activity and conformational stability. We used Cryo-EM to determine the structures of full-length human TH without and with DA, and the structure of S40 phosphorylated TH, complemented with biophysical and biochemical characterizations and molecular dynamics simulations. TH presents a tetrameric structure with dimerized regulatory domains that are separated 15 Å from the catalytic domains. Upon DA binding, a 20-residue α-helix in the flexible N-terminal tail of the regulatory domain is fixed in the active site, blocking it, while S40-phosphorylation forces its egress. The structures reveal the molecular basis of the inhibitory and stabilizing effects of DA and its counteraction by S40-phosphorylation, key regulatory mechanisms for homeostasis of DA and TH., (© 2022. The Author(s).)
- Published
- 2022
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187. Combining Electron Microscopy (EM) and Cross-Linking Mass Spectrometry (XL-MS) for Structural Characterization of Protein Complexes.
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Quintana-Gallardo L, Maestro-López M, Martín-Benito J, Marcilla M, Rutz D, Buchner J, Valpuesta JM, and Cuéllar J
- Subjects
- Molecular Biology, Proteins, Cryoelectron Microscopy, Mass Spectrometry
- Abstract
Structural biology has recently witnessed the benefits of the combined use of two complementary techniques: electron microscopy (EM) and cross-linking mass spectrometry (XL-MS). EM (especially its cryogenic variant cryo-EM) has proven to be a very powerful tool for the structural determination of proteins and protein complexes, even at an atomic level. In a complementary way, XL-MS allows the precise characterization of particular interactions when residues are located in close proximity. When working from low-resolution, negative-staining images and less-defined regions of flexible domains (whose mapping is made possible by cryo-EM), XL-MS can provide critical information on specific amino acids, thus identifying interacting regions and helping to deduce the overall protein structure. The protocol described here is particularly well suited for the study of protein complexes whose intrinsically flexible or transient nature prevents their high-resolution characterization by any structural technique itself., (© 2022. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)
- Published
- 2022
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188. Arc self-association and formation of virus-like capsids are mediated by an N-terminal helical coil motif.
- Author
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Eriksen MS, Nikolaienko O, Hallin EI, Grødem S, Bustad HJ, Flydal MI, Merski I, Hosokawa T, Lascu D, Akerkar S, Cuéllar J, Chambers JJ, O'Connell R, Muruganandam G, Loris R, Touma C, Kanhema T, Hayashi Y, Stratton MM, Valpuesta JM, Kursula P, Martinez A, and Bramham CR
- Subjects
- Animals, Capsid Proteins genetics, Cryoelectron Microscopy, Crystallography, X-Ray, Cytoskeletal Proteins genetics, Drosophila Proteins ultrastructure, Humans, Nerve Tissue Proteins genetics, Neuronal Plasticity genetics, Protein Domains genetics, RNA genetics, Sequence Homology, Amino Acid, Signal Transduction genetics, Virion genetics, Amino Acid Motifs genetics, Cytoskeletal Proteins ultrastructure, Drosophila Proteins genetics, Nerve Tissue Proteins ultrastructure, Neurons metabolism, Protein Conformation
- Abstract
Activity-regulated cytoskeleton-associated protein (Arc) is a protein interaction hub with diverse roles in intracellular neuronal signaling, and important functions in neuronal synaptic plasticity, memory, and postnatal cortical development. Arc has homology to retroviral Gag protein and is capable of self-assembly into virus-like capsids implicated in the intercellular transfer of RNA. However, the molecular basis of Arc self-association and capsid formation is largely unknown. Here, we identified a 28-amino-acid stretch in the mammalian Arc N-terminal (NT) domain that is necessary and sufficient for self-association. Within this region, we identified a 7-residue oligomerization motif, critical for the formation of virus-like capsids. Purified wild-type Arc formed capsids as shown by transmission and cryo-electron microscopy, whereas mutant Arc with disruption of the oligomerization motif formed homogenous dimers. An atomic-resolution crystal structure of the oligomerization region peptide demonstrated an antiparallel coiled-coil interface, strongly supporting NT-NT domain interactions in Arc oligomerization. The NT coil-coil interaction was also validated in live neurons using fluorescence lifetime FRET imaging, and mutation of the oligomerization motif disrupted Arc-facilitated endocytosis. Furthermore, using single-molecule photobleaching, we show that Arc mRNA greatly enhances higher-order oligomerization in a manner dependent on the oligomerization motif. In conclusion, a helical coil in the Arc NT domain supports self-association above the dimer stage, mRNA-induced oligomerization, and formation of virus-like capsids. DATABASE: The coordinates and structure factors for crystallographic analysis of the oligomerization region were deposited at the Protein Data Bank with the entry code 6YTU., (© 2020 Federation of European Biochemical Societies.)
- Published
- 2021
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189. Engineering protein assemblies with allosteric control via monomer fold-switching.
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Campos LA, Sharma R, Alvira S, Ruiz FM, Ibarra-Molero B, Sadqi M, Alfonso C, Rivas G, Sanchez-Ruiz JM, Romero Garrido A, Valpuesta JM, and Muñoz V
- Subjects
- Allosteric Regulation, Cloning, Molecular, Molecular Docking Simulation, Molecular Dynamics Simulation, Mutation, Protein Structure, Secondary genetics, Proteins genetics, Proteins isolation & purification, Recombinant Proteins genetics, Recombinant Proteins isolation & purification, Recombinant Proteins metabolism, Serine Proteases metabolism, Serine Proteinase Inhibitors genetics, Serine Proteinase Inhibitors isolation & purification, Protein Engineering methods, Protein Folding, Protein Multimerization genetics, Proteins metabolism, Serine Proteinase Inhibitors metabolism
- Abstract
The macromolecular machines of life use allosteric control to self-assemble, dissociate and change shape in response to signals. Despite enormous interest, the design of nanoscale allosteric assemblies has proven tremendously challenging. Here we present a proof of concept of allosteric assembly in which an engineered fold switch on the protein monomer triggers or blocks assembly. Our design is based on the hyper-stable, naturally monomeric protein CI2, a paradigm of simple two-state folding, and the toroidal arrangement with 6-fold symmetry that it only adopts in crystalline form. We engineer CI2 to enable a switch between the native and an alternate, latent fold that self-assembles onto hexagonal toroidal particles by exposing a favorable inter-monomer interface. The assembly is controlled on demand via the competing effects of temperature and a designed short peptide. These findings unveil a remarkable potential for structural metamorphosis in proteins and demonstrate key principles for engineering protein-based nanomachinery.
- Published
- 2019
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190. Molecular architecture of the Bardet-Biedl syndrome protein 2-7-9 subcomplex.
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Ludlam WG, Aoba T, Cuéllar J, Bueno-Carrasco MT, Makaju A, Moody JD, Franklin S, Valpuesta JM, and Willardson BM
- Subjects
- Bardet-Biedl Syndrome metabolism, Cilia metabolism, HEK293 Cells, Humans, Mass Spectrometry methods, Microscopy, Electron methods, Models, Molecular, Mutation, Adaptor Proteins, Signal Transducing metabolism, Cytoskeletal Proteins metabolism, Proteins metabolism
- Abstract
Bardet-Biedl syndrome (BBS) is a genetic disorder characterized by malfunctions in primary cilia resulting from mutations that disrupt the function of the BBSome, an 8-subunit complex that plays an important role in protein transport in primary cilia. To better understand the molecular basis of BBS, here we used an integrative structural modeling approach consisting of EM and chemical cross-linking coupled with MS analyses, to analyze the structure of a BBSome 2-7-9 subcomplex consisting of three homologous BBS proteins, BBS2, BBS7, and BBS9. The resulting molecular model revealed an overall structure that resembles a flattened triangle. We found that within this structure, BBS2 and BBS7 form a tight dimer through a coiled-coil interaction and that BBS9 associates with the dimer via an interaction with the α-helical domain of BBS2. Interestingly, a BBS-associated mutation of BBS2 (R632P) is located in its α-helical domain at the interface between BBS2 and BBS9, and binding experiments indicated that this mutation disrupts the BBS2-BBS9 interaction. This finding suggests that BBSome assembly is disrupted by the R632P substitution, providing molecular insights that may explain the etiology of BBS in individuals harboring this mutation., (© 2019 Ludlam et al.)
- Published
- 2019
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191. Elevated levels of Secreted-Frizzled-Related-Protein 1 contribute to Alzheimer's disease pathogenesis.
- Author
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Esteve P, Rueda-Carrasco J, Inés Mateo M, Martin-Bermejo MJ, Draffin J, Pereyra G, Sandonís Á, Crespo I, Moreno I, Aso E, Garcia-Esparcia P, Gomez-Tortosa E, Rábano A, Fortea J, Alcolea D, Lleo A, Heneka MT, Valpuesta JM, Esteban JA, Ferrer I, Domínguez M, and Bovolenta P
- Subjects
- ADAM10 Protein biosynthesis, ADAM10 Protein genetics, Alzheimer Disease pathology, Amyloid Precursor Protein Secretases biosynthesis, Amyloid Precursor Protein Secretases genetics, Amyloid beta-Protein Precursor genetics, Animals, Antibodies, Blocking therapeutic use, Brain Chemistry genetics, Down-Regulation, Humans, Long-Term Potentiation, Membrane Proteins antagonists & inhibitors, Membrane Proteins biosynthesis, Mice, Mice, Transgenic, Neurites pathology, Plaque, Amyloid drug therapy, Plaque, Amyloid genetics, Plaque, Amyloid pathology, Alzheimer Disease genetics, Alzheimer Disease metabolism, Intercellular Signaling Peptides and Proteins genetics, Intercellular Signaling Peptides and Proteins metabolism, Membrane Proteins genetics, Membrane Proteins metabolism
- Abstract
The deposition of aggregated amyloid-β peptides derived from the pro-amyloidogenic processing of the amyloid precurson protein (APP) into characteristic amyloid plaques (APs) is distinctive to Alzheimer's disease (AD). Alternative APP processing via the metalloprotease ADAM10 prevents amyloid-β formation. We tested whether downregulation of ADAM10 activity by its secreted endogenous inhibitor secreted-frizzled-related protein 1 (SFRP1) is a common trait of sporadic AD. We demonstrate that SFRP1 is significantly increased in the brain and cerebrospinal fluid of patients with AD, accumulates in APs and binds to amyloid-β, hindering amyloid-β protofibril formation. Sfrp1 overexpression in an AD-like mouse model anticipates the appearance of APs and dystrophic neurites, whereas its genetic inactivation or the infusion of α-SFRP1-neutralizing antibodies favors non-amyloidogenic APP processing. Decreased Sfrp1 function lowers AP accumulation, improves AD-related histopathological traits and prevents long-term potentiation loss and cognitive deficits. Our study unveils SFRP1 as a crucial player in AD pathogenesis and a promising AD therapeutic target.
- Published
- 2019
- Full Text
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192. Structural and functional analysis of the role of the chaperonin CCT in mTOR complex assembly.
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Cuéllar J, Ludlam WG, Tensmeyer NC, Aoba T, Dhavale M, Santiago C, Bueno-Carrasco MT, Mann MJ, Plimpton RL, Makaju A, Franklin S, Willardson BM, and Valpuesta JM
- Subjects
- Amino Acid Sequence, Binding Sites, Cryoelectron Microscopy, Gene Expression Regulation physiology, Gene Knockdown Techniques, HEK293 Cells, Hep G2 Cells, Humans, Mass Spectrometry, Models, Molecular, Protein Binding, Protein Conformation, Protein Folding, Regulatory-Associated Protein of mTOR genetics, TOR Serine-Threonine Kinases genetics, mTOR Associated Protein, LST8 Homolog genetics, Chaperonin Containing TCP-1 physiology, Regulatory-Associated Protein of mTOR metabolism, TOR Serine-Threonine Kinases metabolism, mTOR Associated Protein, LST8 Homolog metabolism
- Abstract
The mechanistic target of rapamycin (mTOR) kinase forms two multi-protein signaling complexes, mTORC1 and mTORC2, which are master regulators of cell growth, metabolism, survival and autophagy. Two of the subunits of these complexes are mLST8 and Raptor, β-propeller proteins that stabilize the mTOR kinase and recruit substrates, respectively. Here we report that the eukaryotic chaperonin CCT plays a key role in mTORC assembly and signaling by folding both mLST8 and Raptor. A high resolution (4.0 Å) cryo-EM structure of the human mLST8-CCT intermediate isolated directly from cells shows mLST8 in a near-native state bound to CCT deep within the folding chamber between the two CCT rings, and interacting mainly with the disordered N- and C-termini of specific CCT subunits of both rings. These findings describe a unique function of CCT in mTORC assembly and a distinct binding site in CCT for mLST8, far from those found for similar β-propeller proteins.
- Published
- 2019
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193. Structure and Function of the Cochaperone Prefoldin.
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Arranz R, Martín-Benito J, and Valpuesta JM
- Subjects
- Archaea, Eukaryotic Cells, Molecular Chaperones chemistry, Protein Folding
- Abstract
Molecular chaperones are key players in proteostasis, the balance between protein synthesis, folding, assembly and degradation. They are helped by a plethora of cofactors termed cochaperones, which direct chaperones towards any of these different, sometime opposite pathways. One of these is prefoldin (PFD), present in eukaryotes and in archaea, a heterohexamer whose best known role is the assistance to group II chaperonins (the Hsp60 chaperones found in archaea and the eukaryotic cytosolic) in the folding of proteins in the cytosol, in particular cytoskeletal proteins. However, over the last years it has become evident a more complex role for this cochaperone, as it can adopt different oligomeric structures, form complexes with other proteins and be involved in many other processes, both in the cytosol and in the nucleus, different from folding. This review intends to describe the structure and the many functions of this interesting macromolecular complex.
- Published
- 2018
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194. Structural characterization of the NAP; the major adhesion complex of the human pathogen Mycoplasma genitalium.
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Scheffer MP, Gonzalez-Gonzalez L, Seybert A, Ratera M, Kunz M, Valpuesta JM, Fita I, Querol E, Piñol J, Martín-Benito J, and Frangakis AS
- Subjects
- Bacterial Adhesion genetics, Mycoplasma genetics, Mycoplasma metabolism, Mycoplasma Infections microbiology, Mycoplasma genitalium genetics, Mycoplasma genitalium ultrastructure, Organelles, Urethritis microbiology, Bacterial Adhesion physiology, Mycoplasma genitalium metabolism
- Abstract
Mycoplasma genitalium, the causative agent of non-gonococcal urethritis and pelvic inflammatory disease in humans, is a small eubacterium that lacks a peptidoglycan cell wall. On the surface of its plasma membrane is the major surface adhesion complex, known as NAP that is essential for adhesion and gliding motility of the organism. Here, we have performed cryo-electron tomography of intact cells and detergent permeabilized M. genitalium cell aggregates, providing sub-tomogram averages of free and cell-attached NAPs respectively, revealing a tetrameric complex with two-fold rotational (C2) symmetry. Each NAP has two pairs of globular lobes (named α and β lobes), arranged as a dimer of heterodimers with each lobe connected by a stalk to the cell membrane. The β lobes are larger than the α lobes by 20%. Classification of NAPs showed that the complex can tilt with respect to the cell membrane. A protein complex containing exclusively the proteins P140 and P110, was purified from M. genitalium and was structurally characterized by negative-stain single particle EM reconstruction. The close structural similarity found between intact NAPs and the isolated P140/P110 complexes, shows that dimers of P140/P110 heterodimers are the only components of the extracellular region of intact NAPs in M. genitalium., (© 2017 John Wiley & Sons Ltd.)
- Published
- 2017
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195. Clathrin-coat disassembly illuminates the mechanisms of Hsp70 force generation.
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Sousa R, Liao HS, Cuéllar J, Jin S, Valpuesta JM, Jin AJ, and Lafer EM
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- Amino Acid Sequence, Animals, Binding Sites, Cryoelectron Microscopy, Entropy, HSP72 Heat-Shock Proteins chemistry, Hydrogen-Ion Concentration, Models, Molecular, Particle Size, Protein Domains, Protein Stability, Protein Structure, Quaternary, Rats, Clathrin Heavy Chains chemistry, HSP72 Heat-Shock Proteins physiology, Protein Multimerization
- Abstract
Hsp70s use ATP hydrolysis to disrupt protein-protein associations and to move macromolecules. One example is the Hsc70- mediated disassembly of the clathrin coats that form on vesicles during endocytosis. Here, we exploited the exceptional features of these coats to test three models-Brownian ratchet, power-stroke and entropic pulling-proposed to explain how Hsp70s transform their substrates. Our data rule out the ratchet and power-stroke models and instead support a collision-pressure mechanism whereby collisions between clathrin-coat walls and Hsc70s drive coats apart. Collision pressure is the complement to the pulling force described in the entropic pulling model. We also found that self-association augments collision pressure, thereby allowing disassembly of clathrin lattices that have been predicted to be resistant to disassembly. These results illuminate how Hsp70s generate the forces that transform their substrates.
- Published
- 2016
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196. High resolution atomic force microscopy of double-stranded RNA.
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Ares P, Fuentes-Perez ME, Herrero-Galán E, Valpuesta JM, Gil A, Gomez-Herrero J, and Moreno-Herrero F
- Subjects
- Mechanical Phenomena, Microscopy, Atomic Force, RNA, Double-Stranded chemistry
- Abstract
Double-stranded (ds) RNA mediates the suppression of specific gene expression, it is the genetic material of a number of viruses, and a key activator of the innate immune response against viral infections. The ever increasing list of roles played by dsRNA in the cell and its potential biotechnological applications over the last decade has raised an interest for the characterization of its mechanical properties and structure, and that includes approaches using Atomic Force Microscopy (AFM) and other single-molecule techniques. Recent reports have resolved the structure of dsDNA with AFM at unprecedented resolution. However, an equivalent study with dsRNA is still lacking. Here, we have visualized the double helix of dsRNA under near-physiological conditions and at sufficient resolution to resolve the A-form sub-helical pitch periodicity. We have employed different high-sensitive force-detection methods and obtained images with similar spatial resolution. Therefore, we show here that the limiting factors for high-resolution AFM imaging of soft materials in liquid medium are, rather than the imaging mode, the force between the tip and the sample and the sharpness of the tip apex.
- Published
- 2016
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197. Identification of Key Amino Acid Residues Modulating Intracellular and In vitro Microcin E492 Amyloid Formation.
- Author
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Aguilera P, Marcoleta A, Lobos-Ruiz P, Arranz R, Valpuesta JM, Monasterio O, and Lagos R
- Abstract
Microcin E492 (MccE492) is a pore-forming bacteriocin produced and exported by Klebsiella pneumoniae RYC492. Besides its antibacterial activity, excreted MccE492 can form amyloid fibrils in vivo as well as in vitro. It has been proposed that bacterial amyloids can be functional playing a biological role, and in the particular case of MccE492 it would control the antibacterial activity. MccE492 amyloid fibril's morphology and formation kinetics in vitro have been well-characterized, however, it is not known which amino acid residues determine its amyloidogenic propensity, nor if it forms intracellular amyloid inclusions as has been reported for other bacterial amyloids. In this work we found the conditions in which MccE492 forms intracellular amyloids in Escherichia coli cells, that were visualized as round-shaped inclusion bodies recognized by two amyloidophilic probes, 2-4'-methylaminophenyl benzothiazole and thioflavin-S. We used this property to perform a flow cytometry-based assay to evaluate the aggregation propensity of MccE492 mutants, that were designed using an in silico prediction of putative aggregation hotspots. We established that the predicted amino acid residues 54-63, effectively act as a pro-amyloidogenic stretch. As in the case of other amyloidogenic proteins, this region presented two gatekeeper residues (P57 and P59), which disfavor both intracellular and in vitro MccE492 amyloid formation, preventing an uncontrolled aggregation. Mutants in each of these gatekeeper residues showed faster in vitro aggregation and bactericidal inactivation kinetics, and the two mutants were accumulated as dense amyloid inclusions in more than 80% of E. coli cells expressing these variants. In contrast, the MccE492 mutant lacking residues 54-63 showed a significantly lower intracellular aggregation propensity and slower in vitro polymerization kinetics. Electron microscopy analysis of the amyloids formed in vitro by these mutants revealed that, although with different efficiency, all formed fibrils morphologically similar to wild-type MccE492. The physiological implication of MccE492 intracellular amyloid formation is probably similar to the inactivation process observed for extracellular amyloids, and could be used as a mean of sequestering potentially toxic species inside the cell when this bacteriocin is produced in large amounts.
- Published
- 2016
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198. Electron microscopy: the coming of age of a structural biology technique.
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Valpuesta JM and Carrascosa JL
- Subjects
- Microscopy, Electron methods, Molecular Biology methods
- Published
- 2015
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199. Mechano-chemical kinetics of DNA replication: identification of the translocation step of a replicative DNA polymerase.
- Author
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Morin JA, Cao FJ, Lázaro JM, Arias-Gonzalez JR, Valpuesta JM, Carrascosa JL, Salas M, and Ibarra B
- Subjects
- Biological Transport, Kinetics, DNA Replication, DNA-Directed DNA Polymerase metabolism
- Abstract
During DNA replication replicative polymerases move in discrete mechanical steps along the DNA template. To address how the chemical cycle is coupled to mechanical motion of the enzyme, here we use optical tweezers to study the translocation mechanism of individual bacteriophage Phi29 DNA polymerases during processive DNA replication. We determine the main kinetic parameters of the nucleotide incorporation cycle and their dependence on external load and nucleotide (dNTP) concentration. The data is inconsistent with power stroke models for translocation, instead supports a loose-coupling mechanism between chemical catalysis and mechanical translocation during DNA replication. According to this mechanism the DNA polymerase works by alternating between a dNTP/PPi-free state, which diffuses thermally between pre- and post-translocated states, and a dNTP/PPi-bound state where dNTP binding stabilizes the post-translocated state. We show how this thermal ratchet mechanism is used by the polymerase to generate work against large opposing loads (∼50 pN)., (© The Author(s) 2015. Published by Oxford University Press on behalf of Nucleic Acids Research.)
- Published
- 2015
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200. Structures of the Gβ-CCT and PhLP1-Gβ-CCT complexes reveal a mechanism for G-protein β-subunit folding and Gβγ dimer assembly.
- Author
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Plimpton RL, Cuéllar J, Lai CW, Aoba T, Makaju A, Franklin S, Mathis AD, Prince JT, Carrascosa JL, Valpuesta JM, and Willardson BM
- Subjects
- Amino Acids metabolism, Animals, Benzophenones, Carrier Proteins ultrastructure, Chaperonin Containing TCP-1 ultrastructure, Cross-Linking Reagents metabolism, Cryoelectron Microscopy, GTP-Binding Protein beta Subunits ultrastructure, GTP-Binding Protein gamma Subunits ultrastructure, Humans, Mass Spectrometry, Models, Molecular, Nerve Tissue Proteins ultrastructure, Phenylalanine analogs & derivatives, Protein Structure, Secondary, Carrier Proteins chemistry, Chaperonin Containing TCP-1 chemistry, GTP-Binding Protein beta Subunits chemistry, GTP-Binding Protein gamma Subunits chemistry, Nerve Tissue Proteins chemistry, Protein Multimerization
- Abstract
G-protein signaling depends on the ability of the individual subunits of the G-protein heterotrimer to assemble into a functional complex. Formation of the G-protein βγ (Gβγ) dimer is particularly challenging because it is an obligate dimer in which the individual subunits are unstable on their own. Recent studies have revealed an intricate chaperone system that brings Gβ and Gγ together. This system includes cytosolic chaperonin containing TCP-1 (CCT; also called TRiC) and its cochaperone phosducin-like protein 1 (PhLP1). Two key intermediates in the Gβγ assembly process, the Gβ-CCT and the PhLP1-Gβ-CCT complexes, were isolated and analyzed by a hybrid structural approach using cryo-electron microscopy, chemical cross-linking coupled with mass spectrometry, and unnatural amino acid cross-linking. The structures show that Gβ interacts with CCT in a near-native state through interactions of the Gγ-binding region of Gβ with the CCTγ subunit. PhLP1 binding stabilizes the Gβ fold, disrupting interactions with CCT and releasing a PhLP1-Gβ dimer for assembly with Gγ. This view provides unique insight into the interplay between CCT and a cochaperone to orchestrate the folding of a protein substrate.
- Published
- 2015
- Full Text
- View/download PDF
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