191 results on '"Valpuesta, José M."'
Search Results
152. Biochemical Characterization of Symmetric GroEL-GroES Complexes
- Author
-
Llorca, Oscar, primary, Carrascosa, José L., additional, and Valpuesta, José M., additional
- Published
- 1996
- Full Text
- View/download PDF
153. The formation of symmetrical GroEL-GroES complexes in the presence of ATP
- Author
-
Llorca, Oscar, primary, Marco, Sergio, additional, Carrascosa, José L., additional, and Valpuesta, José M., additional
- Published
- 1994
- Full Text
- View/download PDF
154. DNA conformational change induced by the bacteriophage Φ29 connector
- Author
-
Valpuesta, José M., primary, Serrano, Manuel, additional, Donate, Luis E., additional, Herranz, Lucía, additional, and Carrascosa, José L., additional
- Published
- 1992
- Full Text
- View/download PDF
155. Mechanical stability of low-humidity single DNA molecules.
- Author
-
Hormeño, Silvia, Ibarra, Borja, Valpuesta, José M., Carrascosa, José L., and Ricardo Arias-Gonzalez, J.
- Abstract
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]
- Published
- 2012
- Full Text
- View/download PDF
156. Crystal structure of the open conformation of the mammalian chaperonin CCT in complex with tubulin.
- Author
-
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
- Subjects
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]
- Published
- 2011
- Full Text
- View/download PDF
157. The structure of CCT–Hsc70NBD suggests a mechanism for Hsp70 delivery of substrates to the chaperonin.
- Author
-
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.
- Subjects
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]
- Published
- 2008
- Full Text
- View/download PDF
158. Three-Dimensional Structure of Cloned T3 Connector Protein at 1.6nm Resolution
- Author
-
Carrascosa, José L., primary, Valpuesta, José M., additional, and Fujisawa, Hisao, additional
- Published
- 1990
- Full Text
- View/download PDF
159. Structure of eukaryotic prefoldin and of its complexes with unfolded actin and the cytosolic chaperonin CCT.
- Author
-
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.
- Subjects
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]
- Published
- 2002
- Full Text
- View/download PDF
160. The 'sequential allosteric ring' mechanism in the eukaryotic chaperonin-assisted folding of actin and tubulin.
- Author
-
Llorca, Oscar, Martín-Benito, Jaime, Grantham, Julie, Ritco-Vonsovici, Monica, Willison, Keith R., Carrascosa, José L., and Valpuesta, José M.
- Subjects
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]
- Published
- 2001
- Full Text
- View/download PDF
161. Eukaryotic chaperonin CCT stabilizes actin and tubulin folding intermediates in open quasi-native conformations.
- Author
-
Llorca, Oscar, Martín-Benito, Jaime, Ritco-Vonsovici, Monica, Grantham, Julie, Hynes, Gillian M., Willison, Keith R., Carrascosa, José L., and Valpuesta, José M.
- Subjects
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]
- Published
- 2000
- Full Text
- View/download PDF
162. Synthesis of Mesoporous Silica Coated Gold Nanorods Loaded with Methylene Blue and Its Potentials in Antibacterial Applications.
- Author
-
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
- Subjects
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]
- Published
- 2021
- Full Text
- View/download PDF
163. 3D reconstruction of the ATP-bound form of CCT reveals the asymmetric folding conformation of a type II chaperonin.
- Author
-
Llorca, Oscar, Smyth, Martin G., Carrascosa, José L., Willison, Keith R., Radermacher, Michael, Steinbacher, Stefan, and Valpuesta, José M.
- Subjects
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.
- Author
-
Urbaneja, Maíra A., Rivas, Susana, Carrascosa, José L., and Valpuesta, José M.
- Subjects
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]
- Published
- 1994
- Full Text
- View/download PDF
165. Infrared spectroscopic studies of detergent-solubilized uncoupling protein from brown-adipose-tissue mitochondria.
- Author
-
Rial, Eduardo, Muga, Arturo, Valpuesta, José M., Arrondo, José-Luis R., and Goñi, Félix M.
- Subjects
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
- Full Text
- View/download PDF
166. Structural mechanism for tyrosine hydroxylase inhibition by dopamine and reactivation by Ser40 phosphorylation
- Author
-
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]
- Subjects
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.
- Published
- 2022
167. Assisted assembly of bacteriophage T7 core components for genome translocation across the bacterial envelope
- Author
-
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]
- Subjects
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
- Author
-
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.
- Published
- 1992
- Full Text
- View/download PDF
169. Structural insights into the ability of nucleoplasmin to assemble and chaperone histone octamers for DNA deposition
- Author
-
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
- Author
-
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]
- Subjects
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
- Author
-
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
- Subjects
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)
- Published
- 2021
172. Combining Electron Microscopy (EM) and Cross-Linking Mass Spectrometry (XL-MS) for Structural Characterization of Protein Complexes.
- Author
-
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
- Full Text
- View/download PDF
173. Electron microscopy: the coming of age of a structural biology technique.
- Author
-
Valpuesta JM and Carrascosa JL
- Subjects
- Microscopy, Electron methods, Molecular Biology methods
- Published
- 2015
- Full Text
- View/download PDF
174. Mechano-chemical kinetics of DNA replication: identification of the translocation step of a replicative DNA polymerase.
- Author
-
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
- Full Text
- View/download PDF
175. Structures of the Gβ-CCT and PhLP1-Gβ-CCT complexes reveal a mechanism for G-protein β-subunit folding and Gβγ dimer assembly.
- Author
-
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
176. Phosphoinositide 3-kinase beta protects nuclear envelope integrity by controlling RCC1 localization and Ran activity.
- Author
-
Redondo-Muñoz J, Pérez-García V, Rodríguez MJ, Valpuesta JM, and Carrera AC
- Subjects
- Animals, Cell Cycle, Chromatin metabolism, Class I Phosphatidylinositol 3-Kinases, Fibroblasts metabolism, HEK293 Cells, Humans, Lipid Bilayers, Mice, Microscopy, Confocal, Microscopy, Electron, NIH 3T3 Cells, Protein Binding, Cell Cycle Proteins metabolism, Guanine Nucleotide Exchange Factors metabolism, Nuclear Envelope metabolism, Nuclear Proteins metabolism, Phosphatidylinositol 3-Kinases metabolism, ran GTP-Binding Protein metabolism
- Abstract
The nuclear envelope (NE) forms a barrier between the nucleus and the cytosol that preserves genomic integrity. The nuclear lamina and nuclear pore complexes (NPCs) are NE components that regulate nuclear events through interaction with other proteins and DNA. Defects in the nuclear lamina are associated with the development of laminopathies. As cells depleted of phosphoinositide 3-kinase beta (PI3Kβ) showed an aberrant nuclear morphology, we studied the contribution of PI3Kβ to maintenance of NE integrity. pik3cb depletion reduced the nuclear membrane tension, triggered formation of areas of lipid bilayer/lamina discontinuity, and impaired NPC assembly. We show that one mechanism for PI3Kβ regulation of NE/NPC integrity is its association with RCC1 (regulator of chromosome condensation 1), the activator of nuclear Ran GTPase. PI3Kβ controls RCC1 binding to chromatin and, in turn, Ran activation. These findings suggest that PI3Kβ regulates the nuclear envelope through upstream regulation of RCC1 and Ran., (Copyright © 2015, American Society for Microbiology. All Rights Reserved.)
- Published
- 2015
- Full Text
- View/download PDF
177. Structural characterization of the substrate transfer mechanism in Hsp70/Hsp90 folding machinery mediated by Hop.
- Author
-
Alvira S, Cuéllar J, Röhl A, Yamamoto S, Itoh H, Alfonso C, Rivas G, Buchner J, and Valpuesta JM
- Subjects
- HSP40 Heat-Shock Proteins metabolism, HSP70 Heat-Shock Proteins ultrastructure, HSP90 Heat-Shock Proteins ultrastructure, Heat-Shock Proteins ultrastructure, Humans, Microscopy, Electron, Protein Binding, Protein Folding, Protein Structure, Tertiary, Receptors, Glucocorticoid metabolism, HSP70 Heat-Shock Proteins metabolism, HSP90 Heat-Shock Proteins metabolism
- Abstract
In eukarya, chaperones Hsp70 and Hsp90 act coordinately in the folding and maturation of a range of key proteins with the help of several co-chaperones, especially Hop. Although biochemical data define the Hop-mediated Hsp70-Hsp90 substrate transfer mechanism, the intrinsic flexibility of these proteins and the dynamic nature of their complexes have limited the structural studies of this mechanism. Here we generate several complexes in the Hsp70/Hsp90 folding pathway (Hsp90:Hop, Hsp90:Hop:Hsp70 and Hsp90:Hop:Hsp70 with a fragment of the client protein glucocorticoid receptor (GR-LBD)), and determine their 3D structure using electron microscopy techniques. Our results show that one Hop molecule binds to one side of the Hsp90 dimer in both extended and compact conformations, through Hop domain rearrangement that take place when Hsp70 or Hsp70:GR-LBD bind to Hsp90:Hop. The compact conformation of the Hsp90:Hop:Hsp70:GR-LBD complex shows that GR-LBD binds to the side of the Hsp90 dimer opposite the Hop attachment site.
- Published
- 2014
- Full Text
- View/download PDF
178. Molecular determinants of the ATP hydrolysis asymmetry of the CCT chaperonin complex.
- Author
-
Chagoyen M, Carrascosa JL, Pazos F, and Valpuesta JM
- Subjects
- Chaperonin Containing TCP-1 chemistry, Conserved Sequence, Databases, Protein, Hydrolysis, Protein Subunits chemistry, Protein Subunits metabolism, Adenosine Triphosphate metabolism, Chaperonin Containing TCP-1 metabolism, Models, Molecular
- Abstract
The eukaryotic cytosolic chaperonin CCT is a molecular machine involved in assisting the folding of proteins involved in important cellular processes. Like other chaperonins, CCT is formed by a double-ring structure but, unlike all of them, each ring is composed of eight different, albeit homologous subunits. This complexity has probably to do with the specificity in substrate interaction and with the mechanism of protein folding that takes place during the chaperonin functional cycle, but its detailed molecular basis remains unknown. We have analyzed the known proteomes in search of residues that are differentially conserved in the eight subunits, as predictors of functional specificity (specificity-determining positions; SDPs). We have found that most of these SDPs are located near the ATP binding site, and that they define four CCT clusters, corresponding to subunits CCT3, CCT6, CCT8 and CCT1/2/4/5/7. Our results point to a spatial organisation of the CCT subunits in two opposite areas of the ring and provide a molecular explanation for the previously described asymmetry in the hydrolysis of ATP., (Copyright © 2014 Wiley Periodicals, Inc.)
- Published
- 2014
- Full Text
- View/download PDF
179. Programmed cell death protein 5 interacts with the cytosolic chaperonin containing tailless complex polypeptide 1 (CCT) to regulate β-tubulin folding.
- Author
-
Tracy CM, Gray AJ, Cuéllar J, Shaw TS, Howlett AC, Taylor RM, Prince JT, Ahn NG, Valpuesta JM, and Willardson BM
- Subjects
- Apoptosis Regulatory Proteins genetics, Cell Line, Tumor, Chaperonin Containing TCP-1 genetics, Humans, Multiprotein Complexes genetics, Multiprotein Complexes metabolism, Neoplasm Proteins genetics, Protein Structure, Secondary, Protein Structure, Tertiary, Tubulin genetics, Apoptosis Regulatory Proteins metabolism, Chaperonin Containing TCP-1 metabolism, Neoplasm Proteins metabolism, Protein Folding, Tubulin metabolism
- Abstract
Programmed cell death protein 5 (PDCD5) has been proposed to act as a pro-apoptotic factor and tumor suppressor. However, the mechanisms underlying its apoptotic function are largely unknown. A proteomics search for binding partners of phosducin-like protein, a co-chaperone for the cytosolic chaperonin containing tailless complex polypeptide 1 (CCT), revealed a robust interaction between PDCD5 and CCT. PDCD5 formed a complex with CCT and β-tubulin, a key CCT-folding substrate, and specifically inhibited β-tubulin folding. Cryo-electron microscopy studies of the PDCD5·CCT complex suggested a possible mechanism of inhibition of β-tubulin folding. PDCD5 bound the apical domain of the CCTβ subunit, projecting above the folding cavity without entering it. Like PDCD5, β-tubulin also interacts with the CCTβ apical domain, but a second site is found at the sensor loop deep within the folding cavity. These orientations of PDCD5 and β-tubulin suggest that PDCD5 sterically interferes with β-tubulin binding to the CCTβ apical domain and inhibits β-tubulin folding. Given the importance of tubulins in cell division and proliferation, PDCD5 might exert its apoptotic function at least in part through inhibition of β-tubulin folding.
- Published
- 2014
- Full Text
- View/download PDF
180. Yeast mitochondrial RNAP conformational changes are regulated by interactions with the mitochondrial transcription factor.
- Author
-
Drakulic S, Wang L, Cuéllar J, Guo Q, Velázquez G, Martín-Benito J, Sousa R, and Valpuesta JM
- Subjects
- DNA chemistry, Fungal Proteins chemistry, Mitochondria enzymology, Models, Molecular, Promoter Regions, Genetic, Protein Conformation, Transcription Elongation, Genetic, Yeasts enzymology, DNA-Binding Proteins chemistry, DNA-Directed RNA Polymerases chemistry, Mitochondria genetics, Mitochondrial Proteins chemistry, Transcription Factors chemistry, Transcription Initiation, Genetic
- Abstract
Mitochondrial RNA polymerases (MtRNAPs) are members of the single-subunit RNAP family, the most well-characterized member being the RNAP from T7 bacteriophage. MtRNAPs are, however, functionally distinct in that they depend on one or more transcription factors to recognize and open the promoter and initiate transcription, while the phage RNAPs are capable of performing these tasks alone. Since the transcriptional mechanisms that are conserved in phage and mitochondrial RNAPs have been so effectively characterized in the phage enzymes, outstanding structure-mechanism questions concern those aspects that are distinct in the MtRNAPs, particularly the role of the mitochondrial transcription factor(s). To address these questions we have used both negative staining and cryo-EM to generate three-dimensional reconstructions of yeast MtRNAP initiation complexes with and without the mitochondrial transcription factor (MTF1), and of the elongation complex. Together with biochemical experiments, these data indicate that MTF1 uses multiple mechanisms to drive promoter opening, and that its interactions with the MtRNAP regulate the conformational changes undergone by the latter enzyme as it traverses the template strand., (© The Author(s) 2014. Published by Oxford University Press on behalf of Nucleic Acids Research.)
- Published
- 2014
- Full Text
- View/download PDF
181. Mechanical identities of RNA and DNA double helices unveiled at the single-molecule level.
- Author
-
Herrero-Galán E, Fuentes-Perez ME, Carrasco C, Valpuesta JM, Carrascosa JL, Moreno-Herrero F, and Arias-Gonzalez JR
- Subjects
- Microscopy, Atomic Force, DNA chemistry, RNA chemistry
- Abstract
Double-stranded (ds) RNA is the genetic material of a variety of viruses and has been recently recognized as a relevant molecule in cells for its regulatory role. Despite that the elastic response of dsDNA has been thoroughly characterized in recent years in single-molecule stretching experiments, an equivalent study with dsRNA is still lacking. Here, we have engineered long dsRNA molecules for their individual characterization contrasting information with dsDNA molecules of the same sequence. It is known that dsRNA is an A-form molecule unlike dsDNA, which exhibits B-form in physiological conditions. These structural types are distinguished at the single-molecule level with atomic force microscopy (AFM) and are the basis to understand their different elastic response. Force-extension curves of dsRNA with optical and magnetic tweezers manifest two main regimes of elasticity, an entropic regime whose end is marked by the A-form contour-length and an intrinsic regime that ends in a low-cooperative overstretching transition in which the molecule extends to 1.7 times its A-form contour-length. DsRNA does not switch between the A and B conformations in the presence of force. Finally, dsRNA presents both a lower stretch modulus and overstretching transition force than dsDNA, whereas the electrostatic and intrinsic contributions to the persistence length are larger.
- Published
- 2013
- Full Text
- View/download PDF
182. The structure of native influenza virion ribonucleoproteins.
- Author
-
Arranz R, Coloma R, Chichón FJ, Conesa JJ, Carrascosa JL, Valpuesta JM, Ortín J, and Martín-Benito J
- Subjects
- Animals, Cell Nucleus metabolism, Cell Nucleus virology, Cryoelectron Microscopy, Electron Microscope Tomography, Image Processing, Computer-Assisted, Influenza A Virus, H1N1 Subtype physiology, Influenza A Virus, H1N1 Subtype ultrastructure, Madin Darby Canine Kidney Cells, Microscopy, Electron, Models, Molecular, Nucleocapsid Proteins, Protein Conformation, Protein Structure, Secondary, RNA, Viral metabolism, RNA-Binding Proteins chemistry, RNA-Binding Proteins metabolism, RNA-Binding Proteins ultrastructure, RNA-Dependent RNA Polymerase chemistry, RNA-Dependent RNA Polymerase metabolism, RNA-Dependent RNA Polymerase ultrastructure, Ribonucleoproteins metabolism, Ribonucleoproteins ultrastructure, Transcription, Genetic, Viral Core Proteins chemistry, Viral Core Proteins metabolism, Viral Core Proteins ultrastructure, Viral Proteins metabolism, Viral Proteins ultrastructure, Virion ultrastructure, Influenza A Virus, H1N1 Subtype chemistry, RNA, Viral chemistry, Ribonucleoproteins chemistry, Viral Proteins chemistry, Virion chemistry
- Abstract
The influenza viruses cause annual epidemics of respiratory disease and occasional pandemics, which constitute a major public-health issue. The segmented negative-stranded RNAs are associated with the polymerase complex and nucleoprotein (NP), forming ribonucleoproteins (RNPs), which are responsible for virus transcription and replication. We describe the structure of native RNPs derived from virions. They show a double-helical conformation in which two NP strands of opposite polarity are associated with each other along the helix. Both strands are connected by a short loop at one end of the particle and interact with the polymerase complex at the other end. This structure will be relevant for unraveling the mechanisms of nuclear import of parental virus RNPs, their transcription and replication, and the encapsidation of progeny RNPs into virions.
- Published
- 2012
- Full Text
- View/download PDF
183. Active DNA unwinding dynamics during processive DNA replication.
- Author
-
Morin JA, Cao FJ, Lázaro JM, Arias-Gonzalez JR, Valpuesta JM, Carrascosa JL, Salas M, and Ibarra B
- Subjects
- DNA Helicases metabolism, DNA, Viral chemistry, DNA-Directed DNA Polymerase metabolism, Gene Expression Regulation, Viral physiology, Molecular Motor Proteins physiology, Nucleic Acid Conformation, Stress, Mechanical, Viral Proteins genetics, Viral Proteins metabolism, Bacillus Phages genetics, DNA Helicases genetics, DNA Replication genetics, DNA, Viral genetics, DNA-Directed DNA Polymerase genetics
- 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.
- Published
- 2012
- Full Text
- View/download PDF
184. Characterization of the structure and self-recognition of the human centrosomal protein NA14: implications for stability and function.
- Author
-
Rodríguez-Rodríguez M, Treviño MA, Laurents DV, Arranz R, Valpuesta JM, Rico M, Bruix M, and Jiménez MA
- Subjects
- Amino Acid Sequence, Animals, Autoantigens genetics, Centrosome metabolism, Circular Dichroism, Humans, Hydrogen Bonding, Microscopy, Electron, Molecular Sequence Data, Nuclear Magnetic Resonance, Biomolecular, Nuclear Proteins genetics, Protein Conformation, Protein Denaturation, Protein Folding, Protein Multimerization, Protein Structure, Secondary, Sequence Alignment, Solubility, Spectrophotometry, Ultraviolet, Water, Autoantigens chemistry, Nuclear Proteins chemistry
- 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 pK(a) 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.
- Published
- 2011
- Full Text
- View/download PDF
185. SADB phosphorylation of gamma-tubulin regulates centrosome duplication.
- Author
-
Alvarado-Kristensson M, Rodríguez MJ, Silió V, Valpuesta JM, and Carrera AC
- Subjects
- Alternative Splicing genetics, Amino Acid Sequence, Animals, Cell Cycle, Cell Line, Tumor, Gene Expression Regulation, Enzymologic, Gene Knockdown Techniques, Humans, Intracellular Signaling Peptides and Proteins chemistry, Intracellular Signaling Peptides and Proteins genetics, Isoenzymes chemistry, Isoenzymes genetics, Isoenzymes metabolism, Mice, Microtubules enzymology, Molecular Sequence Data, Phosphorylation, Phosphoserine metabolism, Protein Serine-Threonine Kinases chemistry, Protein Serine-Threonine Kinases genetics, Protein Transport, RNA, Messenger genetics, RNA, Messenger metabolism, Tubulin chemistry, Centrosome enzymology, Intracellular Signaling Peptides and Proteins metabolism, Protein Serine-Threonine Kinases metabolism, Tubulin metabolism
- Abstract
Symmetrical cell division requires duplication of DNA and protein content to generate two daughter cells. Centrosomes also duplicate during cell division, but the mechanism controlling this process is incompletely understood. We describe an alternative splice form of SadB encoding a short SADB Ser/Thr kinase whose activity fluctuates during the cell cycle, localizes to centrosomes, and controls centrosome duplication. Reduction of endogenous SADB levels diminished centrosome numbers, whereas enhanced SADB expression induced centrosome amplification. SADB exerted this action through phosphorylation of gamma-tubulin on Ser 131, as expression of a phosphomimetic Ser 131-to-Asp gamma-tubulin mutant alone increased centrosome numbers, whereas non-phosphorylatable Ala 131-gamma-tubulin impaired centrosome duplication. We propose that SADB kinase activity controls centrosome homeostasis by regulating phosphorylation of gamma-tubulin.
- Published
- 2009
- Full Text
- View/download PDF
186. Alpha,gamma-peptide nanotube templating of one-dimensional parallel fullerene arrangements.
- Author
-
Reiriz C, Brea RJ, Arranz R, Carrascosa JL, Garibotti A, Manning B, Valpuesta JM, Eritja R, Castedo L, and Granja JR
- Subjects
- Models, Molecular, Molecular Structure, Nanotubes, Peptide ultrastructure, Fullerenes chemistry, Nanotubes, Peptide chemistry
- Abstract
The formation and full characterization of single self-assembling alpha,gamma-peptide nanotubes (alpha,gamma-SPNs) is described. The introduction of C(60) into cyclic peptides allows the preparation of supramolecular 1D fullerene arrangements induced by peptide nanotube formation under appropriate conditions.
- Published
- 2009
- Full Text
- View/download PDF
187. The structure of CCT-Hsc70 NBD suggests a mechanism for Hsp70 delivery of substrates to the chaperonin.
- Author
-
Cuéllar J, Martín-Benito J, Scheres SH, Sousa R, Moro F, López-Viñas E, Gómez-Puertas P, Muga A, Carrascosa JL, and Valpuesta JM
- Subjects
- Amino Acid Sequence, Animals, Cattle, Chaperonin Containing TCP-1, Escherichia coli Proteins chemistry, HSC70 Heat-Shock Proteins metabolism, Molecular Chaperones chemistry, Molecular Sequence Data, Protein Binding, Protein Conformation, Protein Isoforms, Protein Structure, Tertiary, Sequence Homology, Amino Acid, Chaperonins chemistry, HSP70 Heat-Shock Proteins chemistry
- 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 CCT beta 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.
- Published
- 2008
- Full Text
- View/download PDF
188. All three chaperonin genes in the archaeon Haloferax volcanii are individually dispensable.
- Author
-
Kapatai G, Large A, Benesch JL, Robinson CV, Carrascosa JL, Valpuesta JM, Gowrinathan P, and Lund PA
- Subjects
- Archaeal Proteins genetics, Archaeal Proteins ultrastructure, Chaperonins genetics, Chaperonins ultrastructure, Genes, Archaeal, Genetic Complementation Test, Phenotype, Archaeal Proteins physiology, Chaperonins physiology, Gene Expression Regulation, Archaeal, Haloferax volcanii genetics, Haloferax volcanii growth & development
- 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.
- Published
- 2006
- Full Text
- View/download PDF
189. Structure of the complex between the cytosolic chaperonin CCT and phosducin-like protein.
- Author
-
Martín-Benito J, Bertrand S, Hu T, Ludtke PJ, McLaughlin JN, Willardson BM, Carrascosa JL, and Valpuesta JM
- Subjects
- Amino Acid Sequence, Animals, Carrier Proteins ultrastructure, Cattle, Chaperonin Containing TCP-1, Chaperonins ultrastructure, Microscopy, Electron, Models, Molecular, Molecular Chaperones, Molecular Sequence Data, Multiprotein Complexes chemistry, Multiprotein Complexes metabolism, Multiprotein Complexes ultrastructure, Nerve Tissue Proteins ultrastructure, Protein Binding, Protein Structure, Quaternary, Protein Subunits chemistry, Protein Subunits metabolism, Rats, Sequence Alignment, Carrier Proteins chemistry, Carrier Proteins metabolism, Chaperonins chemistry, Chaperonins metabolism, Nerve Tissue Proteins chemistry, Nerve Tissue Proteins metabolism
- 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 CCT activity, has been solved by cryoelectron microscopy. Binding of PhLP to CCT 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 PhLP 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.
- Published
- 2004
- Full Text
- View/download PDF
190. Molecular clamp mechanism of substrate binding by hydrophobic coiled-coil residues of the archaeal chaperone prefoldin.
- Author
-
Lundin VF, Stirling PC, Gomez-Reino J, Mwenifumbo JC, Obst JM, Valpuesta JM, and Leroux MR
- Subjects
- Archaeal Proteins chemistry, Archaeal Proteins isolation & purification, Binding Sites, Chromatography, Gel, Kinetics, Molecular Chaperones chemistry, Molecular Chaperones isolation & purification, Mutagenesis, Site-Directed, Point Mutation, Polymerase Chain Reaction, Protein Conformation, Recombinant Fusion Proteins chemistry, Recombinant Fusion Proteins isolation & purification, Recombinant Fusion Proteins metabolism, Archaeal Proteins metabolism, Molecular Chaperones metabolism
- 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.
- Published
- 2004
- Full Text
- View/download PDF
191. 3D structure of the influenza virus polymerase complex: localization of subunit domains.
- Author
-
Area E, Martín-Benito J, Gastaminza P, Torreira E, Valpuesta JM, Carrascosa JL, and Ortín J
- Subjects
- Image Processing, Computer-Assisted, Influenza A virus genetics, Microscopy, Electron, Models, Molecular, Protein Structure, Tertiary, Protein Subunits, RNA-Dependent RNA Polymerase genetics, RNA-Dependent RNA Polymerase ultrastructure, Ribonucleoproteins chemistry, Ribonucleoproteins genetics, Ribonucleoproteins ultrastructure, Viral Proteins genetics, Viral Proteins ultrastructure, Influenza A virus enzymology, RNA-Dependent RNA Polymerase chemistry, Viral Proteins chemistry
- 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.
- Published
- 2004
- Full Text
- View/download PDF
Catalog
Discovery Service for Jio Institute Digital Library
For full access to our library's resources, please sign in.