RESUMEN
The molecular chaperone and heat shock protein Hsp90 is part of many protein complexes in eukaryotic cells. Together with its cochaperones, Hsp90 is responsible for the maturation of hundreds of clients. Although having been investigated for decades, it still is largely unknown which components are necessary for a functional complex and how the energy of ATP hydrolysis is used to enable cyclic operation. Here we use single-molecule FRET to show how cochaperones introduce directionality into Hsp90's conformational changes during its interaction with the client kinase Ste11. Three cochaperones are needed to couple ATP turnover to these conformational changes. All three are therefore essential for a functional cyclic operation, which requires coupling to an energy source. Finally, our findings show how the formation of sub-complexes in equilibrium followed by a directed selection of the functional complex can be the most energy efficient pathway for kinase maturation.
Asunto(s)
Proteínas HSP90 de Choque Térmico , Chaperonas Moleculares , Humanos , Hidrólisis , Proteínas HSP90 de Choque Térmico/metabolismo , Chaperonas Moleculares/metabolismo , Adenosina Trifosfato/metabolismo , Unión ProteicaRESUMEN
Protein dynamics have been investigated on a wide range of time scales. Nano- and picosecond dynamics have been assigned to local fluctuations, while slower dynamics have been attributed to larger conformational changes. However, it is largely unknown how fast (local) fluctuations can lead to slow global (allosteric) changes. Here, fast molecule-spanning dynamics on the 100 to 200 ns time scale in the heat shock protein 90 (Hsp90) are shown. Global real-space movements are assigned to dynamic modes on this time scale, which is possible by a combination of single-molecule fluorescence, quasi-elastic neutron scattering and all-atom molecular dynamics (MD) simulations. The time scale of these dynamic modes depends on the conformational state of the Hsp90 dimer. In addition, the dynamic modes are affected to various degrees by Sba1, a co-chaperone of Hsp90, depending on the location within Hsp90, which is in very good agreement with MD simulations. Altogether, this data is best described by fast molecule-spanning dynamics, which precede larger conformational changes in Hsp90 and might be the molecular basis for allostery. This integrative approach provides comprehensive insights into molecule-spanning dynamics on the nanosecond time scale for a multi-domain protein.
Asunto(s)
Proteínas HSP90 de Choque Térmico , Simulación de Dinámica Molecular , Conformación Proteica , Proteínas HSP90 de Choque Térmico/metabolismo , Chaperonas Moleculares/metabolismoRESUMEN
The heat shock protein 90 (Hsp90) is a molecular chaperone, which plays a key role in eukaryotic protein homeostasis. Co-chaperones assist Hsp90 in client maturation and in regulating essential cellular processes such as cell survival, signal transduction, gene regulation, hormone signaling, and neurodegeneration. Aha1 (activator of Hsp90 ATPase) is a unique co-chaperone known to stimulate the ATP hydrolysis of Hsp90, but the mechanism of their interaction is still unclear. In this report, we show that one or two Aha1 molecules can bind to one Hsp90 dimer and that the binding stoichiometry affects Hsp90's conformation, kinetics, ATPase activity, and stability. In particular, a coordination of two Aha1 molecules can be seen in stimulating the ATPase activity of Hsp90 and the unfolding of the middle domain, whereas the conformational equilibrium and kinetics are hardly affected by the stoichiometry of bound Aha1. Altogether, we show a regulation mechanism through the stoichiometry of Aha1 going far beyond a regulation of Hsp90's conformation.
Asunto(s)
Proteínas HSP90 de Choque Térmico , Chaperonas Moleculares , Humanos , Chaperonas Moleculares/metabolismo , Proteínas HSP90 de Choque Térmico/metabolismo , Adenosina Trifosfatasas/metabolismo , Conformación MolecularRESUMEN
Heat shock protein 90 (Hsp90) is a molecular chaperone that assists protein folding in an Adenosine triphosphate (ATP)-dependent way. Hsp90 has been reported to interact with Alzheimers disease associated amyloid-ß (Aß) peptides and to suppress toxic oligomer- and fibril formation. However, the mechanism remains largely unclear. Here we use a combination of atomic force microscopy (AFM) imaging, circular dichroism (CD) spectroscopy and biochemical analysis to quantify this interaction and put forward a microscopic picture including rate constants for the different transitions towards fibrillation. We show that Hsp90 binds to Aß40 monomers weakly but inhibits Aß40 from growing into fibrils at substoichiometric concentrations. ATP impedes this interaction, presumably by modulating Hsp90's conformational dynamics and reducing its hydrophobic surface. Altogether, these results might indicate alternative ways to prevent Aß40 fibrillation by manipulating chaperones that are already abundant in the brain.
Asunto(s)
Adenosina Trifosfato/química , Péptidos beta-Amiloides/química , Amiloide/química , Proteínas HSP90 de Choque Térmico/química , Fragmentos de Péptidos/química , Adenosina Trifosfato/metabolismo , Amiloide/metabolismo , Péptidos beta-Amiloides/metabolismo , Proteínas HSP90 de Choque Térmico/metabolismo , Interacciones Hidrofóbicas e Hidrofílicas , Microscopía de Fuerza Atómica , Modelos Moleculares , Fragmentos de Péptidos/metabolismo , Agregado de Proteínas , Unión Proteica , Conformación Proteica , Proteínas Recombinantes , Análisis Espectral , Relación Estructura-ActividadRESUMEN
Histone lysine methylation is generally performed by SET domain methyltransferases and regulates chromatin structure and gene expression. Here, we identify human C21orf127 (HEMK2, N6AMT1, PrmC), a member of the seven-ß-strand family of putative methyltransferases, as a novel histone lysine methyltransferase. C21orf127 functions as an obligate heterodimer with TRMT112, writing the methylation mark on lysine 12 of histone H4 (H4K12) in vitro and in vivo. We characterized H4K12 recognition by solving the crystal structure of human C21orf127-TRMT112, hereafter termed 'lysine methyltransferase 9' (KMT9), in complex with S-adenosyl-homocysteine and H4K12me1 peptide. Additional analyses revealed enrichment for KMT9 and H4K12me1 at the promoters of numerous genes encoding cell cycle regulators and control of cell cycle progression by KMT9. Importantly, KMT9 depletion severely affects the proliferation of androgen receptor-dependent, as well as that of castration- and enzalutamide-resistant prostate cancer cells and xenograft tumors. Our data link H4K12 methylation with KMT9-dependent regulation of androgen-independent prostate tumor cell proliferation, thereby providing a promising paradigm for the treatment of castration-resistant prostate cancer.
Asunto(s)
Proliferación Celular/fisiología , Histonas/metabolismo , Lisina/metabolismo , Neoplasias de la Próstata Resistentes a la Castración/patología , Metiltransferasa de ADN de Sitio Específico (Adenina Especifica)/metabolismo , Línea Celular Tumoral , Dimerización , Histonas/química , Humanos , Masculino , Metilación , Metiltransferasas/química , Metiltransferasas/metabolismo , Metiltransferasa de ADN de Sitio Específico (Adenina Especifica)/química , Metiltransferasa de ADN de Sitio Específico (Adenina Especifica)/fisiologíaRESUMEN
Members of the multihaem cytochrome c family such as pentahaem cytochrome c nitrite reductase (NrfA) or octahaem hydroxylamine oxidoreductase (Hao) are involved in various microbial respiratory electron transport chains. Some members of the Hao subfamily, here called εHao proteins, have been predicted from the genomes of nitrate/nitrite-ammonifying bacteria that usually lack NrfA. Here, εHao proteins from the host-associated Epsilonproteobacteria Campylobacter fetus and Campylobacter curvus and the deep-sea hydrothermal vent bacteria Caminibacter mediatlanticus and Nautilia profundicola were purified as εHao-maltose binding protein fusions produced in Wolinella succinogenes. All four proteins were able to catalyze reduction of nitrite (yielding ammonium) and hydroxylamine whereas hydroxylamine oxidation was negligible. The introduction of a tyrosine residue at a position known to cause covalent trimerization of Hao proteins did neither stimulate hydroxylamine oxidation nor generate the Hao-typical absorbance maximum at 460 nm. In most cases, the εHao-encoding gene haoA was situated downstream of haoC, which predicts a tetrahaem cytochrome c of the NapC/NrfH family. This suggested the formation of a membrane-bound HaoCA assembly reminiscent of the menaquinol-oxidizing NrfHA complex. The results indicate that εHao proteins form a subfamily of ammonifying cytochrome c nitrite reductases that represents a 'missing link' in the evolution of NrfA and Hao enzymes.
Asunto(s)
Citocromos c/metabolismo , Oxidorreductasas/metabolismo , Proteínas Bacterianas/metabolismo , Grupo Citocromo c , Citocromos a1/metabolismo , Citocromos c1/metabolismo , Epsilonproteobacteria/genética , Epsilonproteobacteria/metabolismo , Nitrato Reductasas/metabolismo , Nitritos/metabolismo , Oxidación-Reducción , Oxidorreductasas/genética , Wolinella/genéticaRESUMEN
Phloroglucinol reductases (PGRs) are involved in anaerobic degradation in bacteria, in which they catalyze the dearomatization of phloroglucinol into dihydrophloroglucinol. We identified three PGRs, from different bacterial species, that are members of the family of NAD(P)H-dependent short-chain dehydrogenases/reductases (SDRs). In addition to catalyzing the reduction of the physiological substrate, the three enzymes exhibit activity towards 2,4,6-trihydroxybenzaldehyde, 2,4,6-trihydroxyacetophenone, and methyl 2,4,6-trihydroxybenzoate. Structural elucidation of PGRcl and comparison to known SDRs revealed a high degree of conservation. Several amino acid positions were identified as being conserved within the PGR subfamily and might be involved in substrate differentiation. The results enable the enzymatic dearomatization of monoaromatic phenol derivatives and provide insight into the functional diversity that may be found in families of enzymes displaying a high degree of structural homology.
Asunto(s)
Bacterias/enzimología , Oxidorreductasas actuantes sobre Donantes de Grupo CH-CH/química , Oxidorreductasas actuantes sobre Donantes de Grupo CH-CH/metabolismo , Acetofenonas/metabolismo , Bacterias/química , Bacterias/metabolismo , Benzaldehídos/metabolismo , Biocatálisis , Ácido Gálico/análogos & derivados , Ácido Gálico/metabolismo , Conformación Proteica , Especificidad por SustratoRESUMEN
The six-electron reduction of sulfite to sulfide is the pivot point of the biogeochemical cycle of the element sulfur. The octahaem cytochrome c MccA (also known as SirA) catalyses this reaction for dissimilatory sulfite utilization by various bacteria. It is distinct from known sulfite reductases because it has a substantially higher catalytic activity and a relatively low reactivity towards nitrite. The mechanistic reasons for the increased efficiency of MccA remain to be elucidated. Here we show that anoxically purified MccA exhibited a 2- to 5.5-fold higher specific sulfite reductase activity than the enzyme isolated under oxic conditions. We determined the three-dimensional structure of MccA to 2.2 Å resolution by single-wavelength anomalous dispersion. We find a homotrimer with an unprecedented fold and haem arrangement, as well as a haem bound to a CX15CH motif. The heterobimetallic active-site haem 2 has a Cu(I) ion juxtaposed to a haem c at a Fe-Cu distance of 4.4 Å. While the combination of metals is reminiscent of respiratory haem-copper oxidases, the oxidation-labile Cu(I) centre of MccA did not seem to undergo a redox transition during catalysis. Intact MccA tightly bound SO2 at haem 2, a dehydration product of the substrate sulfite that was partially turned over due to photoreduction by X-ray irradiation, yielding the reaction intermediate SO. Our data show the biometal copper in a new context and function and provide a chemical rationale for the comparatively high catalytic activity of MccA.
Asunto(s)
Proteínas Bacterianas/química , Cobre/metabolismo , Hemo/análogos & derivados , Oxidorreductasas actuantes sobre Donantes de Grupos Sulfuro/química , Wolinella/enzimología , Proteínas Bacterianas/aislamiento & purificación , Proteínas Bacterianas/metabolismo , Biocatálisis , Dominio Catalítico , Cristalografía por Rayos X , Cisteína/análogos & derivados , Cisteína/metabolismo , Hemo/metabolismo , Modelos Moleculares , Oxidación-Reducción , Oxidorreductasas actuantes sobre Donantes de Grupos Sulfuro/aislamiento & purificación , Oxidorreductasas actuantes sobre Donantes de Grupos Sulfuro/metabolismo , Sulfitos/metabolismo , Dióxido de Azufre/metabolismoRESUMEN
Carbohydrate-based sensors, that specifically detect sugar binding molecules or cells, are increasingly important in medical diagnostic and drug screening. Here we demonstrate that cantilever arrays functionalized with different mannosides allow the real-time detection of several Escherichia coli strains in solution. Cantilever deflection is thereby dependent on the bacterial strain studied and the glycan used as the sensing molecule. The cantilevers exhibit specific and reproducible deflection with a sensitivity range over four orders of magnitude.
Asunto(s)
Carga Bacteriana/instrumentación , Técnicas Biosensibles/instrumentación , Escherichia coli/clasificación , Escherichia coli/aislamiento & purificación , Manósidos/química , Análisis por Micromatrices/instrumentación , Polisacáridos/química , Diseño de Equipo , Análisis de Falla de Equipo , Sistemas Microelectromecánicos/instrumentaciónRESUMEN
Bacterial MCCs (multihaem cytochromes c) represent widespread respiratory electron-transfer proteins. In addition, some of them convert substrates such as nitrite, hydroxylamine, nitric oxide, hydrazine, sulfite, thiosulfate or hydrogen peroxide. In many cases, only a single function is assigned to a specific MCC in database entries despite the fact that an MCC may accept various substrates, thus making it a multifunctional catalyst that can play diverse physiological roles in bacterial respiration, detoxification and stress defence mechanisms. The present article briefly reviews the structure, function and biogenesis of selected MCCs that catalyse key reactions in the biogeochemical nitrogen and sulfur cycles.
Asunto(s)
Bacterias/metabolismo , Biocatálisis , Citocromos c/metabolismo , Hemo/metabolismo , Nitrógeno/metabolismo , Azufre/metabolismo , Ciclo del NitrógenoRESUMEN
Carbohydrates are important mediators of many biological processes that underlie cellular communication and disease mechanisms. Therapeutic agents include carbohydrate-based vaccines and the potent anti-viral protein Cyanovirin-N (CV-N). CV-N acts by specifically binding the carbohydrate structures decorating the cell surface of deadly viruses including human immunodeficiency virus (HI-V) or Ebola. In search for new carbohydrate-binding proteins and the development of sensors that exploit carbohydrate-protein interactions the label-free cantilever array technique can provides a fast, parallel and low-cost approach.
Asunto(s)
Proteínas Bacterianas/análisis , Técnicas Biosensibles/métodos , Carbohidratos/química , Proteínas Portadoras/análisis , Nanotecnología/métodos , Escherichia coli/química , Microscopía de Fuerza Atómica , Polisacáridos/químicaRESUMEN
Advances in carbohydrate sequencing technologies have revealed the tremendous complexity of the glycome. This complexity reflects the structural and chemical diversity of carbohydrates and is greater than that of proteins and oligonucleotides. The next step in understanding the biological function of carbohydrates requires the identification and quantification of carbohydrate interactions with other biomolecules, in particular, with proteins. To this end, we have developed a cantilever array biosensor with a self-assembling carbohydrate-based sensing layer that selectively and sensitively detects carbohydrate-protein binding interactions. Specifically, we examined binding of mannosides and the protein cyanovirin-N, which binds and blocks the human immunodeficiency virus (HIV). Cyanovirin-N binding to immobilized oligomannosides on the cantilever resulted in mechanical surface stress that is transduced into a mechanical force and cantilever bending. The degree and duration of cantilever deflection correlates with the interaction's strength, and comparative binding experiments reveal molecular binding preferences. This study establishes that carbohydrate-based cantilever biosensors are a robust, label-free, and scalable means to analyze carbohydrate-protein interactions and to detect picomolar concentrations of carbohydrate-binding proteins.
Asunto(s)
Carbohidratos/química , Sistemas Microelectromecánicos/instrumentación , Microquímica/instrumentación , Nanotecnología/instrumentación , Mapeo de Interacción de Proteínas/instrumentación , Proteínas/química , Diseño de Equipo , Análisis de Falla de Equipo , Unión ProteicaRESUMEN
An interaction-site model can a priori predict molecular self-organisation on a new substrate in Monte Carlo simulations. This is experimentally confirmed with scanning tunnelling microscopy on Fréchet dendrons of a pentacontane template. Local and global ordering motifs, inclusion molecules and a rotated unit cell are correctly predicted.
RESUMEN
The evaporation of solutions of dendron-functionalised 2,2'-bipyridines on a graphite surface gives highly ordered monolayers; near atomic resolution STM imaging has allowed a detailed conformational analysis to be made.